WO2011132801A1 - Nucleic acid base analog with quenching characteristics and fluorescence and application thereof - Google Patents

Nucleic acid base analog with quenching characteristics and fluorescence and application thereof Download PDF

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WO2011132801A1
WO2011132801A1 PCT/JP2011/060343 JP2011060343W WO2011132801A1 WO 2011132801 A1 WO2011132801 A1 WO 2011132801A1 JP 2011060343 W JP2011060343 W JP 2011060343W WO 2011132801 A1 WO2011132801 A1 WO 2011132801A1
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base
group
formula
artificial
fluorescence
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PCT/JP2011/060343
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French (fr)
Japanese (ja)
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一郎 平尾
路子 平尾
横山　茂之
雅雄 三井
りえ 山重
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独立行政法人理化学研究所
タグシクス・バイオ株式会社
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Priority to EP11772132.4A priority Critical patent/EP2562255A4/en
Priority to JP2012511735A priority patent/JP5874152B2/en
Priority to US13/642,111 priority patent/US9285319B2/en
Publication of WO2011132801A1 publication Critical patent/WO2011132801A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/044—Pyrrole radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer

Definitions

  • the present invention relates to quenching and fluorescent nucleobase analogs and their applications. Specifically, the present invention relates to the discovery and utilization of 2-nitropyrrole and its modified 1- and 4-positions, and their nucleoside derivatives as quenching molecules or nucleobase analogs. Yes, it can be used for a wide range of detection and diagnosis such as visualization of PCR products.
  • the technology for artificially creating new base pairs and extending DNA genetic information is considered to have two highly versatile applications, and research and development of artificial base pairs has been actively conducted.
  • One application is to create artificial base pairs that function in replication, transcription, and translation to create DNA, RNA, and proteins that incorporate new components.
  • Another application is the formation of double-stranded nucleic acids in which artificial base pairs are incorporated into DNA or RNA, thereby increasing the number of nucleic acid fragment probe sequence species to apply to real-time PCR multiplexes and DNA computers. It can be used as a new codon and anticodon when introducing artificial amino acids into proteins by translation.
  • nucleobase analogues exhibiting fluorescence have been reported, but no substance has been known so far that the nucleobase analogue itself exhibits a strong quenching action.
  • a method of binding a quenching molecule such as a dabsyl group to a nucleobase via a linker is known.
  • the base forming the base pair is not completely in contact with the fluorescent molecule located in the vicinity, so that the quenching action is weakened, and its detection is impossible without relying on an apparatus.
  • a simple and efficient detection method for base pairs utilizing the quenching action of bases has not been found.
  • the present inventors have found a base exhibiting quenching properties, and if a base exhibiting fluorescence and a base exhibiting quenching properties selectively form a base pair, when DNA forms a double strand, a fluorescent artificial base is formed.
  • the present inventors have conceived that the present invention has been conceived because it is possible that a base can be strongly quenched, and that a detection technique that can be visually judged by DNA amplification or molecular beacons in PCR can be created.
  • the 2-nitropyrrole derivative is a base represented by Pn or Px, which is an artificial base pair that the present inventors have developed so far.
  • Pn and Px are used as a third nucleic acid base pair (artificial base pair) as a complementary artificial base (Ds: 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group) and a base pair ( Ds-Pn, Ds-Px base pairs), and it is shown that they can be introduced into specific sites in nucleic acids by replication or transcription.
  • 2-nitropyrrole forms a base pair with Dss (7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridine), which is an improved base of Ds, as a fluorescent artificial base. To do.
  • the inventors of the present invention revealed for the first time that 2-nitropyrrole has a quenching property in the present invention. For example, it was found that when Pn or Px forms a base pair with Dss in double-stranded DNA, the fluorescence of Dss is quenched by the quenching property of 2-nitropyrrole. In addition, although 3-nitropyrrole similar to 2-nitropyrrole is known as a universal base, the quenching action is low unlike 2-nitropyrrole of the present invention.
  • the quenching Pn (or Px) is in contact with the fluorescent Dss as a base pair, so that the fluorescence of Dss is efficiently quenched.
  • the double-stranded structure is denatured into single-stranded DNA, a DNA strand containing Dss emits light.
  • a new molecular beacon detection / diagnosis technique has become possible.
  • the fluorescence of the nucleoside or nucleotide derivative is quenched to some extent by the interaction of 2-nitropyrrole and the dye.
  • the substrate (nucleoside triphosphate) of the derivative of Px can be introduced into DNA by duplication, complementing the artificial base Ds in the template.
  • R 1 and R 2 are ribose, deoxyribose, Hydrogen, hydroxyl group, SH group, halogen, A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group, One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms, Sugar, sugar chain, amino acid, peptide, A group independently selected from the group consisting of fluorescent molecules linked via a linker.
  • R 1 is ribose or deoxyribose.
  • a method for detecting the formation of an artificial base pair 1) Formula II
  • R 2 is Hydrogen, hydroxyl group, SH group, halogen, A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group, One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms, Sugar, sugar chain, amino acid, peptide, A fluorescent molecule bound via a linker, A group selected from the group consisting of] A nucleoside or nucleotide having a quenching artificial base represented by: 2) Either a natural base modified with self-quenching, an artificial base, or a nucleoside or nucleotide having a base analog that can be a donor such as fluorescence resonance energy transfer (FRET) or static quenching, Alternatively, both methods are used.
  • FRET fluorescence resonance energy transfer
  • R 2 is Hydrogen, hydroxyl group, SH group, halogen, A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group, One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms, Sugar, sugar chain, amino acid, peptide, A fluorescent molecule bound via a linker, A group selected from the group consisting of] The method as described above, wherein the formation of an artificial base pair is detected by observing a decrease in fluorescence of the fluorescent artificial base by forming a base pair with the quenching artificial base represented by formula (1).
  • a method for detecting the formation of a base pair of an artificial base by lowering the fluorescence of a fluorescent artificial base comprising: (I) 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss); (Ii) 7- (2,2 ′, 5 ′, 2 ′′ -tertien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dsss); (Iii) 2-amino-6- (2,2′-bithien-5-yl) purin-9-yl group (ss); (Iv) 2-amino-6- (2,2 ′, 5 ′, 2 ′′ -tert-en-5-yl) purin-9-yl group (sss); (V) 4- (2,2′-bithien-5-yl) -pyrrolo [2,3-b] pyridin-1-yl group (Dsas
  • R 3 is -H, iodine, -CH 3,
  • R 4 is -CH 3, -CH 2 -NH 2 and,
  • a nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base; and A quenching base of the following formula III or formula IV
  • R 3 is -H, iodine, -CH 3,
  • R 4 is -CH 3, -CH 2 -NH 2 and,
  • R 5 is a fluorescent molecule bound via a linker
  • the method wherein the fluorescence intensity of the fluorescent molecule in the quenching artificial base represented by formula (1) is changed by the formation of the base pair by the artificial base of Formula V, and the formation of the artificial base pair is detected.
  • Fluorescent molecules include indocarbocyanine (Cy3), indodicarbocyanine (Cy5), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA) ), 6-carboxytetramethylrhodamine (6-TAMRA), 5-dimethylaminonaphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (5-HEX), 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (6-HEX), 5-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (5-TET), 6-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (6-TET), 6-carboxy-2
  • a nucleic acid primer comprising a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base; and Base of formula VI:
  • R 6 is a fluorescent molecule bound through or without a linker], and detects the formation of an artificial base base pair by a change in fluorescence intensity. Kit for use in the method.
  • a method for detecting the formation of an artificial base pair Using a nucleic acid containing a polynucleoside having a natural base modifier, an artificial base, or a base analog having a self-quenching property that can serve as a donor such as fluorescence resonance energy transfer (FRET) and static quenching (static quenching), When an artificial base pair is formed between an artificial base (first artificial base) in the nucleic acid and an artificial base having a fluorescent molecule (second artificial base), the modified natural base, artificial base, or base Detects the formation of an artificial base pair by changing the fluorescence spectrum due to fluorescence resonance energy transfer or static quenching from a polynucleoside having an analog to a fluorescent molecule of a second artificial base Said method.
  • FRET fluorescence resonance energy transfer
  • static quenching static
  • R 6 when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed, Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI by excitation with ultraviolet light at 240-390 nm It is detected that the fluorescence spectrum is changed due to static quenching or the like, and an artificial base pair is formed.
  • at least one polynucleotide having a 2-amino-6- (2-thienyl) purin-9-yl group (s) as a base is present on the same strand as a nucleic acid containing a polynucleoside having Ds as a base.
  • R 6 when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed, Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI upon excitation with UV light at 350-390 nm The fluorescence spectrum is changed by static quenching or the like, and it is detected that an artificial base pair is formed.
  • a polynucleotide having a base in which (Dss) is bound to a natural base Said method.
  • Fluorescent substances include indocarbocyanine (Cy3), indodicarbocyanine (Cy5), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA) ), 6-carboxytetramethylrhodamine (6-TAMRA), 5-dimethylaminonaphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (5-HEX), 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (6-HEX), 5-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (5-TET), 6-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (6-TET), 6-carboxy-2
  • nucleic acid primer selected from the group consisting of i) -iii) below: i) a nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base; ii) a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base, and at least one 2-amino-6- (2-thienyl)- A nucleic acid primer comprising a polynucleotide having a 9H-purin-9-yl group (s) as a base; iii) a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base, and at least one
  • R 6 is a fluorescent molecule attached via or without a linker in Formula VI
  • Quencher 1 Quencher Structure
  • the present invention provides a novel quencher.
  • the quencher of the present invention has a 2-nitropyrrole structure represented by Formula I
  • R 1 and R 2 are ribose, deoxyribose, Hydrogen, hydroxyl group, SH group, halogen, A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group, One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms, Sugar, sugar chain, amino acid, peptide, It is a group independently selected from the group consisting of fluorescent molecules linked via a linker. It is characterized by that.
  • R 1 and R 2 are not particularly limited, and any group can be selected.
  • R 1 and R 2 are arbitrarily selected independently.
  • Ribose, deoxyribose R 1 and / or R 2 are preferably ribose or deoxyribose.
  • R 1 is ribose or deoxyribose.
  • “Ribose” is one of pentose monosaccharides, and the IUPAC name is “(3R, 4S, 5R) -5- (hydroxymethyl) tetrahydrofuran-2,3,4-triol”.
  • “Deoxyribose” is a kind of pentose monosaccharide containing an aldehyde group, and the IUPAC name is “(2R, 4S, 5R) -5- (hydroxymethyl) tetrahydrofuran-2,4-diol”. .
  • the partner that forms the base pair or a base that exists in the vicinity is a fluorescent base In some cases, it has a quenching effect that extinguishes the fluorescence. Or the fluorescence of the fluorescent substance couple
  • halogen Hydrogen, hydroxyl group, SH group, halogen
  • the type of halogen is not particularly limited. Preferably, it is selected from the group consisting of fluorine, bromine, iodine and the like.
  • alkyl group, alkenyl group or alkynyl group having 2 to 10 carbon atoms may be linear or branched, and in particular It is not limited.
  • a methyl group, an ethyl group, a propynyl group, an ethylene group, an ethynyl group and the like are included. These groups may be substituted.
  • the substituent is not particularly limited, but is preferably selected from the group consisting of amino group, hydroxyl group, SH group, halogen, carboxyl group, nitro group and the like.
  • one or more 5-membered heterocycles 1 or more 6-membered heterocycles, 1 or more heterocycles, 1 or more aromatic rings
  • R 1 and / or R containing nitrogen or sulfur atoms 2 may be one or more heterocycles.
  • the heterocycle is a 5-membered heterocycle selected from a thienyl group, a thiazolyl group, an imidazolyl group, a furanyl group, or a derivative thereof.
  • 6-membered heterocycles include pyranyl group, pyridyl group, pyrimidyl group, and the like.
  • heterocyclic ring examples include purine, 1-deazapurine, quinoline and the like.
  • aromatic ring examples include a phenyl group and a naphthyl group.
  • the number of heterocycles, heterocycles and aromatic rings is not particularly limited, but is preferably 1 to 3. More preferably, it is 1 or 2.
  • v) Sugar, sugar chain, amino acid, peptide Sugar is not particularly limited. For example, glucose, arabinose, furanose and the like are included. Ribose or deoxyribose is also a kind of sugar.
  • the sugar chain is not particularly limited, and examples thereof include sucrose and lactose.
  • Amino acids are not particularly limited. For example, glycine, alanine, phenylalanine and the like are included.
  • the type of peptide is not particularly limited.
  • the polypeptide consists of about 2 to 10 amino acid residues.
  • a preferred peptide is, for example, phenylalanyl-glycine.
  • non-natural peptides such as peptide nucleic acids are also included.
  • linker is not particularly limited, and those skilled in the art can appropriately employ it.
  • the linker is not limited, but preferably the following chemical formulas VII and VIII:
  • n is selected from an integer of 1 to 12] as well as
  • n, m and l are each independently selected from an integer of 1 to 12] Selected from the group consisting of In formulas VII and VIII, n, m and l are each preferably 1-7, more preferably 5.
  • fluorescent molecule is not particularly limited.
  • Substance having fluorescence to be quenched The type of substance having fluorescence that is quenched by the quenching action of the quencher having a 2-nitropyrrole structure represented by Formula I of the present invention is not particularly limited.
  • Examples of the substance having fluorescence include arbitrary substances such as fluorescent molecules such as fluorescent artificial bases and fluorescent dyes.
  • the 2-nitropyrrole structure represented by the formula I preferably forms a pair with the following base (Japanese Patent Application No. 2009-232851).
  • Dss, Dsss, ss, sss, Dsas, Dsav and Dvas are fluorescent bases.
  • the fluorescence intensity decreases or is quenched.
  • the substance having fluorescence that does not directly form a base pair with the quencher of formula I is present near the artificial base that forms a base pair with the quencher of formula I, Can be affected.
  • an artificial base having fluorescence is present on the same single-stranded or double-stranded or triple-stranded nucleic acid as the above-mentioned artificial base (for example, s) (for example, adjacent to each other), or
  • a substance having fluorescence is formed in the vicinity of the quencher of the present invention by forming a base pair of the artificial base that forms a base pair with the quencher of formula I. Can be affected by quenching.
  • the present invention also provides a method for detecting an artificial base pair.
  • the method of the present invention comprises: 1.
  • R 2 is Hydrogen, hydroxyl group, SH group, halogen, A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group, One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms, Sugar, sugar chain, amino acid, peptide, A fluorescent molecule bound via a linker, A group selected from the group consisting of] A nucleoside or nucleotide having a quenching artificial base represented by: 2) Either a natural base modified with self-quenching, an artificial base, or a nucleoside or nucleotide having a base analog that can be a donor such as fluorescence resonance energy transfer (FRET) or static quenching, Alternatively, both are used.
  • FRET fluorescence resonance energy transfer
  • the nitrogen atom of the pyrrole ring of the quenching artificial base of formula II of the present invention forms a nucleoside or nucleotide by binding to ribose or deoxyribose.
  • the artificial base of the formula II of the present invention forms an artificial base pair with artificial bases such as Ds, Dss, Dsss, s, ss, sss, dDsa, Dsas, Dsav, dDva, Dvas, and dDia (Japanese Patent Application 2009). -232851).
  • the fluorescence intensity of the fluorescent artificial base forming the base pair, or the fluorescent base or fluorescent molecule present in the vicinity changes, or Extinguish.
  • the method of the present invention uses this change to detect the formation of artificial base pairs.
  • Dss, Dsss, ss, sss, Dsas, Dsav and Dvas are fluorescent bases, and by forming a base pair with the compound of formula II, the fluorescence of the artificial base is reduced or quenched. .
  • the fluorescence intensity of the fluorescent molecule is lowered due to the quenching property of the quenching artificial base of the present invention. This is considered to be because the fluorescent molecule is efficiently quenched by stacking with a quenching artificial base in a solution. Then, when the quenching artificial base to which the fluorescent molecule is bound forms an artificial base pair with the artificial base and is incorporated into the nucleic acid, stacking between the fluorescent molecule and the quenching artificial base occurs. Since it is eliminated, the fluorescence intensity of the fluorescent dye increases. Using this property, it is possible to detect the formation of artificial base pairs.
  • the present invention has a self-quenching property that can be a donor such as fluorescence resonance energy transfer (FRET) or static quenching. It also includes a method for detecting an artificial base pair using a nucleoside or nucleotide having a natural base modification, an artificial base, or a base analog.
  • FRET Fluorescence Resonance Energy Transfer
  • FRET Fluorescence resonance energy transfer
  • the method of the present invention utilizes a self-quenching natural base modification, artificial base, or base analog that can be a donor of fluorescence resonance energy transfer (FRET).
  • the quenching process includes static quenching due to the formation of excimers such as excimers in addition to FRET.
  • these natural base modifiers, artificial bases, or base analogs having self-quenching properties are present in the vicinity of the acceptor due to the formation of an artificial base pair, these donors are excited by giving energy of a specific wavelength. As a result, energy is donated from these donors to the acceptor, and the acceptor emits fluorescence with energy of a wavelength that should not emit fluorescence.
  • artificial base having self-quenching includes one or more adjacent s, for example, two or more adjacent s, ss, Dss, and Dsss on the same nucleic acid.
  • “Natural base modified product having self-quenching property” means, but is not limited to, a natural base (for example, uracil to which s is bound, s to 2) to which at least one artificial base having self-quenching properties (for example, s) is bound. Single-linked cytosine, uracil bonded with Dss), and the like.
  • Examples of the “base analog having self-quenching property” include a size-expanded base analog dimer, a 2-aminopurine dimer, and the like.
  • the artificial base pair detected here is preferably a base pair between the quenching artificial base represented by the above formula II and its complementary artificial base. However, it is not necessarily limited thereto. Any method that uses a natural base modifier, an artificial base, or a nucleoside or nucleotide having a base analog that has a self-quenching property that can serve as a donor for fluorescence resonance energy transfer (FRET) or static quenching (static quenching) Other known artificial base pairs are also included in the scope of the present invention.
  • FRET fluorescence resonance energy transfer
  • static quenching static quenching
  • sy base pair (s: 2-amino-6-thienylpurine, y: pyridin-2-one), vy base pair (v: 2-amino-6-thiazolyl purine), s-Pa base pair (Pa: pyrrole-2-carbaldehyde), Ds-Pa base pair (Ds: 7- (2-thienyl) -imidazo [4,5-b] pyridine), Pa-Q base pair (Q: 9-methylimidazo) Artificial base pairs such as [(4,5) -b] pyridine), isoG-isoC, 5SICS-MMO2, and 5NaM can also be detected by the method using FRET of the present invention.
  • FIG. 1 shows an example of an artificial base pair of a quenching base (Pn, Px) and its complementary base (Ds or Dss). Examples are Pn and a fluorescent artificial base (Dss), and an artificial base pair consisting of Px and Ds.
  • FIG. 2 shows the structure of a quenching artificial base Pn and its 4 ′ derivative used in the examples of the present invention.
  • FIG. 3 shows the structures of the quenching artificial base Px and its derivatives used in Examples of the present invention.
  • FIG. 4 shows the structures of Ds and fluorescent artificial bases Dss, Dsss, and Dsav as examples of artificial bases complementary to Pn or Px used in the method of the present invention.
  • FIG. 6 shows the structure of a natural base amidite reagent bound with a fluorescent artificial base Dss or s.
  • the compounds in FIG. 6 are as follows. Among 100mM NaHCO 3, NH 2 -hx- dPxTP (8.4 ⁇ mol), Na 2 CO 3 buffer (pH8.5) (500 ⁇ l), in DMF (300 ⁇ l), Cy3 (7.63 ⁇ mol ) N- hydroxysuccinimide midges Luster, 12 hours at room temperature [FIG. 6]
  • FIG. 6 shows the structure of a natural base amidite reagent bound with a fluorescent artificial base Dss or s. The compounds in FIG.
  • FIG. 6 shows Dss-hx-dU amidite, s-hx-dU amidite, and s2-hx-dC amidite from the left.
  • FIG. 7 shows quenching of the fluorescent artificial base Dss in the complementary strand by the artificial base Pn.
  • Each DNA solution (5 ⁇ M) in 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.1 mM EDAT was photographed by irradiation at 365 nm.
  • FIG. 7 shows the fluorescence spectrum of each DNA fragment in the experiment of FIG.
  • Double-stranded DNA (5′-GGTAACNATGCG-3 ′) containing single-stranded DNA containing Dss (5′-GGTAACDssATGCG-3 ′), Dss-Pn, Dss-Dss, Dss-Ds, and Dss-T base pairs.
  • FIG. 11 shows the results of examining a primer extension reaction using a template DNA containing Pn and dDssTP by Klenow fragment of DNA polymerase I derived from E. coli. Each concentration of dDssTP was added to 200 nM template DNA, 10 ⁇ M dCTP and dTTP, 0.1 U / ⁇ l Klenow fragment, reacted at 37 ° C.
  • FIG. 11 shows that dDssTP is complementary to Pn in the template and incorporated into the complementary strand DNA.
  • FIG. 12 shows the results of examining PCR amplification of DNA containing Ds using Dss-Px base pairs.
  • Ds-containing DNA 55-mer was subjected to 20 cycles of PCR amplification using dDssTP, NH 2 -hx-dPxTP, and a natural base substrate. After denaturing gel electrophoresis, the product was stained with SYBR Green II. Was analyzed.
  • FIG. 13 shows the results of DNA sequencing after PCR amplification using Dss-Px base pairs.
  • DNA (55-mer) containing Ds was subjected to PCR amplification for 15 cycles using dDssTP, NH 2 -hx-dPxTP, and a natural base substrate, and the amplification product was sequenced by a conventional method. It was found that 99% or more of Dss and NH 2 -hx-Px were retained in the amplified DNA.
  • This sequencing uses the method developed by the inventors of the present application (Anna Unusual hydrophobic base pair system: site-specific incorporation of nucleic acids into RNA. , T. Fujiwara, R. Kawai, A. Sato, Y. Harada, S. Yokoyama, Nature Methods, 3, 729-735 (2006), An Unnatural base pir system.
  • FIG. 14 is a schematic diagram showing the principle of real-time PCR using Dss-Px base pairs.
  • Dss is introduced into a PCR primer and PCR is performed using dPnTP or dPxTP
  • Pn and Px are incorporated into the complementary strand in a complementary manner to Dss, and the fluorescence of Dss is quenched. By detecting this, real-time PCR becomes possible.
  • FIG. 15 shows the results of real-time PCR using Dss-Px base pairs.
  • dPxTP is incorporated into its complementary strand, and the fluorescence of Dss is quenched. This forms a Dss-Px base pair during PCR, quenches the fluorescence of Dss, and enables application to real-time PCR.
  • This Dss-Pn (or Dss-Px) base pair property can be applied to molecular beacons.
  • FIG. 17 shows that molecular beacons containing Dss-Pn base pairs can be visualized.
  • the fluorescence of the hairpin beacon (26-mer) containing Dss-Pn base pairs was observed in the presence or absence of the target single-stranded DNA (71-mer).
  • Each DNA was measured in 1 ⁇ M, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA.
  • FIG. 18 shows the result of detecting a single base mutation by a molecular beacon containing Dss-Pn base pairs. The fluorescence of each of the two types of hairpin type beacons (26-mer) containing Dss-Pn base pairs was observed in addition to the single-stranded DNA (71-mer) of each of the two types of target sequences with one base mutation.
  • FIG. 19 shows the principle of visualization PCR using Cy3-Px / Dss base pair as a substrate in which fluorescent dye Cy3 is bound to Px.
  • FIG. 20 shows an example of visual real-time PCR using Cy3-Px / Dss base pairs based on the principle described in FIG.
  • Reaction mixture (25 ⁇ L scale) 1x Titanium Taq PCR buffer 1 ⁇ M 080731-5 ′ primer 3 (SEQ ID NO: 15) 1 ⁇ M 090914a-Plexor-Dss1 (SEQ ID NO: 16) 2 ⁇ M Cy3-hx-dPxTP 2 mM dNTPs 1 ⁇ Titanium TaqDNA polymerase 2aM (3 copies) -200 fM (3000000 copies) Template DNA application The total volume was adjusted to 25 ⁇ L with sterile water.
  • Cy3 does not emit light at an excitation wavelength near 350 nm, and thus Px substrate bound to Cy3 (Cy3-hx-dPxTP) Does not emit light when irradiated with UV at 350 nm.
  • Cy3-hx-dPxTP is incorporated into the complementary strand of Dss due to base pairing of Dss-Px, fluorescence resonance energy transfer (FRET) from Dss to Cy3 occurs due to UV irradiation at 350 to 390 nm, and light is emitted.
  • FRET fluorescence resonance energy transfer
  • FIG. 21 is a schematic diagram showing the principle of a real-time PCR method using a quenching Px base to which a fluorescent molecule (Cy3) is bound.
  • a fluorescent molecule for example, Cy3
  • Cy3-hx-dPxTP a substrate
  • Cy3-hx-dPx a primer into which a Ds base has been introduced
  • Cy3-hx-dPx is incorporated into DNA, thereby increasing the fluorescence intensity of Cy3.
  • FIG. 22 shows the results of real-time PCR using a quenching Px base to which a fluorescent molecule (Cy3) is bound.
  • Real-time PCR detection using Cy3-hx-dPxTP as a substrate was performed using a real-time PCR apparatus (Stratagene, Mx3005P). PCR reaction was performed with primers: 1 ⁇ M each, natural base substrate dNTP: 0.2 mM each, artificial base substrate Cy3-hx-dPxTP: 2 ⁇ M, and a fluorescence change at 568 nm was detected at an excitation wavelength of 545 nm.
  • FIG. 23 shows the result of gel electrophoresis detecting a real-time PCR product with a quenching Px base bound with a fluorescent molecule (Cy3). Since the PCR product shown in FIG.
  • FIG. 24 shows the results of examining the fluorescence characteristics of DNA containing a fluorescent molecule (Cy3) and a fluorescent artificial base s.
  • FIG. 25 shows the principle of a visualization PCR method in which a quenching Px base to which a fluorescent molecule (Cy3) is bound and a fluorescent artificial base s are combined.
  • a quenching Px base to which a fluorescent molecule (Cy3) is bound and a fluorescent artificial base s are combined.
  • Cy3-hx-dPxTP Cy3-hx-Px is incorporated into its complementary strand.
  • FIG. 26 shows the results of a visualization PCR method in which a quenching Px base combined with a fluorescent molecule (Cy3) and a fluorescent artificial base s are combined.
  • FIG. 26 shows the results of performing PCR, illuminating the PCR tube at 350 nm, and examining the light emission of Cy3 with the naked eye or through an orange filter.
  • FIG. 27a shows the results of a visualization PCR method combining a quenching Px base to which a fluorescent molecule (Cy3) is bound and a fluorescent artificial base s.
  • FIG. 27b shows the result of detecting a visualized PCR in which a quenching Px base bound with a fluorescent molecule (Cy3) and a fluorescent artificial base s are combined using a real-time quantitative PCR apparatus.
  • FIG. 27c shows the result of visualizing the DNA amplification product of each PCR cycle of FIG. 27b.
  • FIG. 27d shows the result of quantifying the fluorescence intensity of each PCR tube in FIG. 27c.
  • FIG. 27d1 is a plot of fluorescence intensity in each PCR cycle when 0, 3 to 30000 copies of DNA were amplified by PCR.
  • FIG. 27d2 is a graph plotting the fluorescence intensity when 3-3,000,000 copies of each DNA were amplified in each PCR cycle.
  • FIG. 28 shows the detection of a product on gel electrophoresis by PCR (55 cycles) using a primer in which a quenching Px base bound to a fluorescent molecule (Cy3) and a fluorescent artificial base s are combined.
  • the PCR product By agarose gel electrophoresis of the visualized PCR product of FIG. 27a by the method of the present invention, the PCR product could be detected by irradiation at 312 nm or 532 nm. In the case of irradiation at 312 nm, FRET from s to Cy3 is detected, and in the case of irradiation at 532 nm, the result of direct excitation of Cy3 incorporated in DNA is shown. Since the PCR product is labeled with Cy3, it was possible to observe the PCR product on a gel by exciting s at 312 nm and FRET, or by directly exciting Cy3 at 532 nm. [FIG. 29a] FIG.
  • FIG. 29a uses a nucleoside derivative (FIG. 6, s-hx-dU, (Us)) in which a fluorescent molecule (s base) is bound to a natural base via a linker and a Ds-Px base pair. It is the schematic diagram which showed the detection method of the PCR product which was found. In the visualized PCR method of FIG. 25, two adjacent fluorescent artificial bases s are used. Instead, this fluorescent s is bound to a natural base via a linker, and this base is added to the PCR primer. A configuration in which two are adjacent to each other is the embodiment of FIG. 29a. [FIG. 29b] FIG. 29b shows the use of a nucleoside derivative (FIG.
  • FIG. 29c shows the results of visualization PCR in which a quenching Px base bound with a fluorescent molecule (Cy3) and a fluorescent artificial base s-hx-dU are combined. Real-time PCR is also possible by increasing the fluorescence intensity of Cy3 of Cy-hx-Px incorporated into DNA.
  • FIG. 29d FIG.
  • FIG. 29d is a visualization of the DNA amplification products of each PCR cycle of FIG. 29c.
  • FIG. 30 is a chemical synthesis of s-hx-dU amidite reagent. Conditions: (a) CBr 4 , PPh 3 , CH 2 Cl 2 ; 1. (B) K 2 CO 3 , DMF; 2. (C) Pac-Cl, HOBT, pyridine, CH 3 CN; 3. (D) DMTr-deoxy-5-iodouridine, Pd (PPh 3 ) 4 , CuI, TEA, DMF; 4). (E) NC (CH 2) 2 O-P (Cl) N (iPr) 2, DIEA, THF FIG. 31 shows a nucleoside derivative (FIG.
  • FIG. 32 shows a nucleotide derivative (FIG. 6, Dss-hx-dU (UDss)) in which a fluorescent molecule (Dss base) is bound to a natural base via a linker and a Ds-Px base pair. It is the schematic diagram which showed the detection method of PCR product.
  • FIG. 33 shows the results of chemical synthesis of Dss-hx-dU amidite reagent.
  • the present invention preferably includes the following aspects.
  • the method of the present invention comprises a fluorescent artificial base and a formula II
  • R 2 is Hydrogen, hydroxyl group, SH group, halogen, A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group, One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms, Sugar, sugar chain, amino acid, peptide, A fluorescent molecule bound via a linker, A group selected from the group consisting of] The formation of an artificial base pair is detected by observing a decrease in fluorescence of the fluorescent artificial base by forming a base pair with the quenching artificial base represented by
  • the fluorescent artificial base is known to form base pairs with Formula II: (I) 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss); (Ii) 7- (2,2 ′, 5 ′, 2 ′′ -tertien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dsss); (Iii) 2-amino-6- (2,2′-bithien-5-yl) purin-9-yl group (ss); (Iv) 2-amino-6- (2,2 ′, 5 ′, 2 ′′ -tert-en-5-yl) purin-9-yl group (sss); (V) 4- (2,2′-bithien-5-yl) -pyrrolo [2,3-b] pyridin-1-yl group (Dsas); (Vi) 4- [2- (2-thiazolyl) thien-5-
  • the quenching base of the present invention is of formula II1 or formula IV below.
  • R 3 is -H, iodine, -CH 3,
  • R 4 is -CH 3, -CH 2 -NH 2 and,
  • n is an integer from 0 to 12
  • n is preferably 3-7, more preferably 5.
  • the present invention also provides A nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base; and Provided is a kit for use in a method for detecting the formation of a base pair of an artificial base by reducing the fluorescence of the fluorescent artificial base, comprising a polynucleotide having a quenching base of formula III or formula IV as a base.
  • a method that utilizes the fact that the fluorescence intensity of a fluorescent molecule bound to the quenching artificial base of the present invention changes due to the formation of an artificial base pair is a method for detecting the formation of an artificial base pair.
  • R 5 is a fluorescent molecule bound via a linker
  • Complementary base pairs in which the artificial base of Formula V forms a base pair include any of the above-described Ds, Dss, Dsss, s, ss, sss, dDsa, Dsas, Dsav, dDva, Dvas, and dDia. Is possible. Preferred are Ds, s, ss, sss, dDsa, dDva, and dDia, more preferably 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds).
  • the quenching artificial base is preferably a base of the following formula VI:
  • R 6 is a fluorescent molecule attached via or without a linker.
  • Linkers similar to those described above for the quencher of formula I can be used.
  • Fluorescent molecules can be used similar to those described above for the quencher of formula I.
  • the present invention also provides Ds A nucleic acid primer comprising a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base; and Provided is a kit for use in a method for detecting the formation of an artificial base base pair by a change in fluorescence intensity, comprising a polynucleotide having a base of formula VI.
  • a nucleic acid comprising a natural base modifier having a self-quenching property, an artificial base, or a polynucleoside having a base analog, which can serve as a donor, such as fluorescence resonance energy transfer (FRET) and static quenching (static quenching)
  • FRET fluorescence resonance energy transfer
  • static quenching static quenching
  • a method for detecting the formation of an artificial base pair Utilizing a nucleic acid comprising a natural base modifier having a self-quenching property, an artificial base, or a polynucleoside having a base analog, which can be a donor such as fluorescence resonance energy transfer (FRET) and static quenching,
  • FRET fluorescence resonance energy transfer
  • a nucleic acid comprising a natural base modifier having a self-quenching property, an artificial base, or a polynucleoside having a base analog, which can be a donor such as fluorescence resonance energy transfer (FRET) and static quenching
  • the artificial base pair between an artificial base (first artificial base) in a nucleic acid and an artificial base having a fluorescent molecule (second artificial base) is preferably a quenching artificial base represented by the formula II of the present invention As the second artificial base.
  • a known artificial base pair is a natural base modification having a self-quenching property that can be a donor such as fluorescence resonance energy transfer (FRET) and / or static quenching, It is possible to use a nucleic acid containing a polynucleoside having an artificial base or a base analog.
  • the present invention provides the following aspects as one aspect of the method of C.
  • the method for detecting the formation of a base pair of an artificial base based on the change in the fluorescence spectrum due to the fluorescence resonance energy transfer or static quenching of the present invention is described in 7- (2,2′-biten-5-yl. ) Imidazo [4,5-b] pyridin-3-yl group (Dss) and a base of the following formula VI:
  • FIG. 240-410 nm ultraviolet light is the wavelength at which Dss is excited. It is desirable that the fluorescent molecules in the base of formula VI normally do not fluoresce at this wavelength and that fluorescence is only observed when FRET occurs.
  • an artificial base pair is formed between the 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and the base of formula VI. Is detected.
  • the present invention provides the following aspects as one aspect of the method of C.
  • the method of detecting the base pair formation of an artificial base by the change of the fluorescence spectrum by the fluorescence resonance energy transfer and static quenching of the present invention When a base pair is formed between a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI, Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in the base of formula VI by excitation with ultraviolet light at 240-390 nm Static quenching (static quenching) occurs, the fluorescence spectrum is changed, and it is detected that an artificial base pair is formed.
  • At least one polynucleotide having a 2-amino-6- (2-thienyl) purin-9-yl group (s) as a base is present on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. This is the method.
  • the number of s present on the same strand as the nucleic acid containing a polynucleoside having Ds as a base is not limited, but is preferably 1-3, more preferably 1 or 2. Most preferably 2.
  • lane 3 when the number of s is 2, the fluorescence intensity of s is reduced or quenched due to the self-quenching property of s (self-quenching), and the change in the fluorescence spectrum due to FRET is clearer.
  • s When s is 1, the fluorescence of s is observed (FIG. 24, lane 2). Also in this case, the fluorescence of the fluorescent molecule is observed from the fluorescence of s by FRET (FIG. 24, lanes 5 and 6).
  • the present invention provides the following aspects as one aspect of the method of C.
  • the method for detecting the base pair formation of an artificial base by the change of the fluorescence spectrum by the fluorescence resonance energy transfer or static quenching of the present invention When a base pair is formed between Ds and the Formula VI base, Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI upon excitation with UV light at 350-390 nm Static quenching (static quenching) or the like occurs, and the fluorescence spectrum changes, and it is detected that an artificial base pair is formed.
  • At least one 2-amino-6- (2-thienyl) purin-9-yl group (s) is bonded to a natural base on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. Said method wherein there is at least one polynucleotide having one base.
  • FIGS. 29a and 31 A schematic diagram of this embodiment is shown in FIGS. 29a and 31.
  • the type of natural base to which s binds is not limited, and any of A, T, G, C, and U is possible.
  • the adjacent natural bases may be the same or different.
  • two or more of the same are adjacent.
  • the number of natural bases to which s binds is adjacent to the same nucleic acid as well as the C-2 embodiment in which s is present in the nucleic acid, but it is preferably 1-3, more preferably 1 or 2. is there. Most preferably 2.
  • the mode of C-3 includes a mode in which two or more s are bonded to one natural base (FIG. 31).
  • the number of s to be bonded is not particularly limited, but is preferably 2 or 3, more preferably 2.
  • the present invention provides the following aspects as one aspect of the method of C.
  • the method for detecting the base pair formation of an artificial base by the change of the fluorescence spectrum by the fluorescence resonance energy transfer or static quenching of the present invention When a base pair is formed between Ds and the base of formula VI, Fluorescence in the base of formula VI from 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) upon excitation with 240-410 nm UV light It is detected that the fluorescence spectrum changes due to fluorescence resonance energy transfer or static quenching to the molecule, and an artificial base pair is formed.
  • a polynucleotide having a base in which (Dss) is bound to a natural base Said method.
  • the fluorescent molecule is not particularly limited.
  • the quencher of formula I More preferred is indocarbocyanine (Cy3).
  • the substituent R 6 in the base of formula VI is preferably:
  • the present invention also provides One nucleic acid primer selected from the group consisting of i) -iii) below: i) a nucleic acid primer comprising a polynucleotide having Dss as a base; ii) a nucleic acid primer comprising a polynucleoside having Ds as a base and a polynucleotide having at least one s as a base; iii) a nucleic acid primer comprising a polynucleoside having Ds as a base, and a polynucleotide having at least one s bonded to a natural base, and iv) a polynucleoside having Ds as a base; and A nucleic acid primer comprising a polynucleotide having a base in which Dss is bound to a natural base; And a method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum by flu
  • Dss-Pn and Dss-Px base pairs function efficiently in PCR.
  • the base pair of the nucleic acid may be formed by any step of transcription, reverse transcription, replication or translation.
  • the change in the detection spectrum can be determined with the naked eye.
  • Prior to the present invention there was no visible and simple method for forming an artificial base pair and detecting a target nucleic acid.
  • Real-time PCR can also be visualized using the detection method of the present invention. Therefore, a complicated and expensive PCR apparatus is no longer necessary.
  • nucleic acid amplification is performed using the method for detecting an artificial base pair of the present invention, the amplified nucleic acid is electrophoresed as it is and is easily detected. It is possible (FIG. 23 etc.). Furthermore, quantification is also possible by the intensity of the electrophoresis band.
  • FIG. 8 shows the fluorescence spectrum of each DNA fragment measured using a JASCO FP-6500 spectrometer equipped with an ETC-273T temperature controller.
  • Example 4 Quenching action of Pn (FIG. 9) A. Changes in fluorescence intensity of deoxyribonucleoside triphosphate (dDssTP, 5 ⁇ M) of fluorescent artificial base Dss depending on concentration of deoxyribonucleoside triphosphate (dPnTP) of Pn JASCO FP-6500 spectrometer equipped with ETC-273T temperature controller 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.1 mM EDTA containing 2 mM, 1 mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.05 mM deoxyribonucleoside triphosphate (dPnTP) 5 ⁇ l of deoxyribonucleoside triphosphate (dDssTP, 105 ⁇ M) was added to the solution (100 ⁇ l), and the emission spectrum of dDssTP by excitation at 370 nm was measured at 20 ° C.
  • dDssTP in order to examine the fluorescence quenching effect of dDssTP in the presence of the natural base deoxyribonucleoside triphosphate, 15 mM, 12 mM, 9 mM, 6 mM, 3 mM, 1 mM deoxyriboadenosine triphosphate (dATP), deoxyriboguanosine triphosphate
  • dATP deoxyriboadenosine triphosphate
  • dATP deoxyriboguanosine triphosphate
  • 10 mM sodium phosphate (pH 7.0) 100 mM NaCl, 0.1 mM EDTA solution (100 ⁇ l) containing (dGTP), deoxyribothymidine triphosphate (dTTP), deoxyribocytidine triphosphate (dCTP), deoxyribonucleoside triphosphate 5 ⁇ l of acid (dDssTP, 105 ⁇ M) was added, and the emission spectrum of dDss
  • a quencher (excited at 370 nm) measured in a 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA solution at 20 ° C. (excitation at 370 nm)
  • the Stern-Volmer constant (K SV ) was calculated by substituting the decrease in fluorescence intensity with respect to the concentration of dPnTP, dATP, dGTP, dCTP, dTTP into the following Stern-Volmer equation.
  • FIG. 10 shows the results of measuring fluorescence at a final concentration of 5 ⁇ M dDss in the presence of 5 mM or 5 mM dPn or various derivatives thereof at an excitation wavelength of 385 nm and a measurement temperature of 25 ° C.
  • each nucleoside solution (20 ⁇ M dDss, 20 mM dPn and various derivatives thereof) was prepared by the following procedure.
  • a primer labeled with 6-carboxyfluorescein at the 5 ′ end (20-mer, 5′-ACTCACTATAGGGAGGAAGA-3 ′ (SEQ ID NO: 4) or 5′-ACTCACTATAGGGAGCTTCT-3 ′ (SEQ ID NO: 5))
  • the reaction conditions are summarized as follows. Use 5 ⁇ M primer-template duplex, 5-50 nM enzyme and 0.3-1500 ⁇ M substrate in solution (10 ⁇ l).
  • the solution (10 ⁇ l) contains 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT and 0.05 mg / ml BSA.
  • the reaction is 1-35 minutes at 37 ° C.
  • the Run Module is GS Run 36C-2400. The electrophoresis time was about 1 hour, and the peak pattern of the reaction product was analyzed and quantified with an automatic ABI377 DNA sequencer equipped with GeneScan software (version 3.0).
  • Example 7 Primer extension reaction using Pn-containing template DNA and dDssTP using Klenow fragment of DNA polymerase I derived from E. coli (FIG. 11)
  • Primer (23-mer) (SEQ ID NO: 8) labeled with 32 P at the 5 ′ end and template DNA (35-mer) (SEQ ID NO: 9) containing Pn or Pa, containing 14 mM MgCl 2 and 0.2 mM DTT
  • 20 mM Tris-HCl (pH 7.5) buffer the sample was heated at 95 ° C. and then slowly cooled to 4 ° C. to form a template strand and a primer duplex.
  • nucleoside triphosphate solution (40 ⁇ M dCTP, 40 ⁇ M dTTP, 0-40 ⁇ M dDssTP) was added on ice. .
  • the reaction was started by adding an enzyme solution (2.5 ⁇ l, 1 unit) obtained by diluting Klenow fragment having exonuclease activity (KF exo +, TaKaRa) with sterilized water to this solution. After incubating at 37 ° C.
  • reaction was stopped by adding 1 ⁇ TBE solution (stop solution) containing 10 ⁇ l of 10M urea and heating at 75 ° C. for 3 minutes.
  • stop solution 10 ⁇ l of 10M urea
  • the reaction product was electrophoresed on a 15% polyacrylamide-7 M urea gel, and the band pattern was analyzed by autoradiography using a bioimaging analyzer (FLA7000, Fuji Film).
  • Example 8 PCR amplification of DNA containing Ds using Dss-Px base pairs (FIG. 12)
  • NH 2 -hx-dPxTP and dDssTP template DNA containing Ds (S2, 55-mer) or DNA consisting only of natural bases (control, 55-mer) is used.
  • PCR was performed, and the result of analyzing the product by electrophoresis is shown in FIG.
  • DNA S2 55-mer, underlined primer annealing site
  • DNA control 55-mer, underlined primer annealing site
  • SEQ ID NO: 11 5 'primer: 5'-CGTTGTAAAACGACGGGCCAGGATAATACGACTCACTATAG-3 '(SEQ ID NO: 12) 3 'primer: 5′-TTTCACACAGGAAACAGCTATGAC-3 ′
  • PCR reaction scale: 40 ⁇ l
  • the final composition of the reaction solution was 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, Deep Vent DNA polymerase (0.02 unit / ⁇ l). , NEB), 1 ⁇ M 5 ′ primer, 1 ⁇ M 3 ′ primer, 0.3 mM each natural base substrate dNTP, 10-25 ⁇ M dDssTP, and 25 ⁇ M NH 2 -hx-dPxTP.
  • the PCR product after 20 cycles was electrophoresed on a 15% polyacrylamide-7M urea gel.
  • the amplified DNA band was detected in SYBR mode of Bioimager LAS4000 (Fuji Film) after the gel was stained with SYBR Green II (Lonza).
  • Example 9 Sequencing of DNA after PCR amplification using Dss-Px base pairs (FIG. 13) PCR is performed using a template DNA (S2, 55-mer) containing Ds in the presence of a predetermined concentration of artificial base substrates, NH 2 -hx-dPxTP and dDssTP, and the artificial base Dss is retained in the product.
  • FIG. 13 shows the result of analysis by DNA sequencing using an artificial base substrate dPa′TP or ddPa′TP.
  • DNA S2 55-mer, underlined primer annealing site
  • 5′- TTTCACACAGGAAAACAGCTATGAC GGCCCDsTTGCC CTATAGTGAGTCGTATTATC- 3 ′ (SEQ ID NO: 10)
  • Primer for PCR 5′-side primer: 5′-CGTTGTTAAAACGACGCCCAGGATAATACGACTCACTATAG-3 ′ (SEQ ID NO: 12)
  • 3'-side primer 5'-TTTCACACAGGAAACAGCCTATGAC-3 '(SEQ ID NO: 13)
  • Sequencing primers 5′-CGTTGTTAAAACGACGCCAG-3 ′ (SEQ ID NO: 14)
  • PCR (reaction scale: 25 ⁇ l) was performed using a DNA fragment with a final concentration of 0.6 nM as a template, and 15 cycles were performed with 94 ° C.
  • the final composition of the reaction solution was 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, Deep Vent DNA polymerase (0.02 unit / ⁇ l). NEB), 1 ⁇ M 5 ′ primer, 1 ⁇ M 3 ′ primer, 0.3 mM each natural base substrate dNTP, 2-10 ⁇ M dDssTP, and 2-50 ⁇ M NH 2 -hx-dPxTP.
  • the full length of the PCR product after 15 cycles was purified with a denaturing gel, and then used as a template for a DNA sequence and subjected to sequencing analysis.
  • the DNA sequencing reaction was performed on a commercially available BigDye Terminator v1.1 Cycle Sequencing Kit (Applied BioSystems) with a total volume of 20 ⁇ l, and a PCR amplification fragment (approx. 4 pmol) and about 0.3 pmol of PCR amplified fragment (about 4 pmol). ) And 25 cycles of PCR (96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes) in the presence of 40 pmol of dPa′TP or 1 nmol of ddPa′TP. Unreacted dye terminator was removed from the reaction solution with a Centri-Sep spin column (Applied BioSystems), and the remaining solution was dried by vacuum suction.
  • the residue was suspended by adding 4 ⁇ l of a formamide solution diluted with Blue-Dextran, and a part thereof was analyzed by an ABI377 DNA sequencer.
  • the gel composition used for the analysis was a 7% polyacrylamide-6M urea gel, and the peak pattern of the sequence was analyzed using Applied BioSystems PRISM sequencing analysis v3.2 software.
  • FIG. 14 shows the principle of real-time PCR when PCR is performed in the presence of a dPxTP substrate using a primer containing the artificial base Dss.
  • FIG. 15 shows the results of real-time PCR using the following DNA fragments. A quantitative amplification plot was obtained, and it was possible to detect even 3 copies of DNA in the reaction solution (25 ⁇ l). all right.
  • Sequence used for experiment (underlined part corresponds to primer annealing site) 5′-primer sequence: 5′- CATGTAGATGCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15) 3′-primer sequence: 5′-AATAATGCDss TCCTCAAAGGGTGGACTTC- 3 ′ (SEQ ID NO: 16) Double-stranded template DNA (98 bp; only one strand is listed): 5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17) Specifically, using a real-time PCR apparatus (Stratagene, Mx3005P), in the presence of each primer 1 ⁇ M, natural base substrate dNTP 0.2 mM, artificial base substrate dPxTP 2 ⁇ M, after 94 ° C.
  • the reaction scale of PCR is 25 ⁇ l, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 ⁇ g / ml BSA, 1 ⁇ Titanium Taq DNA polymerase.
  • the DNA fragment used for the template was diluted to 0, 3, 15, 30, 150, 300, 1500, 3000, 15000, 30000 copies in the reaction solution, and PCR was performed for each concentration. .
  • the filter set used for detection is excitation 350 nm-fluorescence 440 nm (for ALEXA).
  • Plexor registered trademark
  • AnalySiS Software v1.5.4.18, Promega & Eragen BioSciences was used. The results are shown in FIG.
  • Example 11 Fluorescence properties of DNA hairpins containing Dss-Pn base pairs (FIG. 16) 2 types of DNA containing Dss, hairpin ssDNA (34-mer) (SEQ ID NO: 18) and ssDNA (12-mer) (SEQ ID NO: 19) become 1 ⁇ M in 1 ⁇ ExTaq Buffer (TaKaRa, containing 2 mM MgCl 2 )
  • a change in fluorescence intensity due to a change in temperature was detected in a dissociation mode of Mx3005P.
  • FIG. 16 shows a graph when the signal is normalized with the value at 35 ° C. after correction with the signal intensity of ROX.
  • the straight-line ssDNA (12-mer) having no structure was a profile in which fluorescence gradually occurred as in the case of only Buffer without DNA (background), whereas Dss-Pn base pair
  • the hairpin ssDNA (34-mer), which forms the hairpin structure that was included, had a profile in which the fluorescence increased with increasing temperature. This is because Pn having a quenching action forms a base pair with Dss because it has a hairpin structure at low temperature, and the fluorescence intensity of Dss is quenched by Pn and the fluorescence intensity is lowered. In this case, the hairpin structure is broken, the quenching action is lost, and the fluorescence of Dss is detected.
  • Example 12 Visualization of molecular beacons using Dss-Pn base pairs (FIG. 17)
  • Various DNA fragments, molecular beacon (MB-C, 26-mer) (SEQ ID NO: 20) and Target DNA (71G, 71-mer) (SEQ ID NO: 21) were prepared to 2 ⁇ M each, and equal amounts (50 ⁇ l each) ) Mixed.
  • a solution containing no Target DNA was mixed with an MB-C solution.
  • the final solution composition is DNA concentration 1 ⁇ M each, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA. This solution was heated at 90 ° C.
  • the molecular beacon In the absence of the target DNA, the molecular beacon forms a loop-stem structure, and the Dss fluorescence is quenched by Dss-Pn base pairing. However, in the presence of the target DNA, the molecular beacon loop part is hybridized. As a result, a double strand was formed with the target DNA, the stem structure was broken, and the Dss-Pn base pair was not formed, and it was confirmed visually that Dss fluorescence was detected.
  • Example 13 Detection of single nucleotide mutation by molecular beacon using Dss-Pn base pair (FIG. 18)
  • a 50 ⁇ l aliquot of a molecular beacon (26-mer, MB-C (SEQ ID NO: 20) or MB-T (SEQ ID NO: 23)) diluted to 500 nM was dispensed, and the Target DNA fragment (71 A sample mixed with -mer, 71G (SEQ ID NO: 21) or 71A (SEQ ID NO: 22), 12.5 ⁇ l) was kept in an incubator at 45 ° C. for 5 minutes or more to obtain an equilibrium state. Fluorescence measurement was performed using a JASCO FP-6500 spectrometer.
  • the composition in the final solution is molecular beacon 400 nM, Target DNA 0-3200 nM, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA.
  • the graph shown in FIG. 18 is plotted after standardizing the fluorescence intensity at 454 nm with the fluorescence intensity in the absence of the Target DNA fragment. It was found that a single base mutation can be detected by a molecular beacon using a Dss-Pn base pair by utilizing the fact that the fluorescence intensity is significantly lower when there is a single base mismatch than when it is a perfect match.
  • FIG. 19 shows the principle of real-time PCR when PCR is performed in the presence of a substrate of Cy3-hx-dPxTP using a primer containing the artificial base Dss.
  • FRET occurs between Dss and Cy3 by irradiation at around 350 nm, and thus PCR-amplified double-stranded DNA can be specifically illuminated. It was also found that the fluorescence by FRET can be detected visually (FIG. 20).
  • sequence used for experiment is the same as FIG. Sequence used in the experiment (underlined part corresponds to the primer annealing site) 5′-primer sequence: 5′- CATGTAGATGCCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15) 3′-primer sequence: 5′-AATAATGCDss TCCTCAAAGGTGGTACTACT- 3 ′ (SEQ ID NO: 16) Double-stranded template DNA (98 bp; only one strand is listed): 5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17) Specifically, using a real-time PCR apparatus (Stratagene, Mx3005P), 94 ° C.-2 minutes in the presence of each primer 1 ⁇ M, natural base substrate dNTP 0.2 mM, artificial base
  • the reaction scale of PCR is 25 ⁇ l, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 ⁇ g / ml BSA, 1 ⁇ Titanium Taq DNA polymerase.
  • the DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration.
  • the reaction tube was directly irradiated with 365 nm UV, and fluorescence was detected visually through an orange filter.
  • FIG. 21 shows the principle of real-time PCR when PCR is performed in the presence of a substrate of a dPxTP derivative to which a fluorescent molecule (such as Cy3) is bound using a primer containing an artificial base Ds.
  • a fluorescent molecule such as Cy3
  • the fluorescence of the fluorescent molecule is quenched by about 30%.
  • this is used as a substrate (Cy3-hx-dPxTP) and PCR is carried out using a primer into which a Ds base has been introduced, Cy3-hx-Px is incorporated into DNA, thereby increasing the fluorescence intensity of Cy3.
  • FIG. 22 shows the results of real-time PCR using the following DNA fragments. A quantitative amplification plot was obtained, and it was possible to detect even 3 copies of DNA in the reaction solution (25 ⁇ l). all right.
  • Sequence used for experiment (underlined part corresponds to primer annealing site) 5′-primer sequence: 5′- CATGTAGATGCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15) 3′-primer sequence: 5′-AATAATGCDs TCCTCAAAGGTGGTACTACT- 3 ′ (SEQ ID NO: 24) Double-stranded template DNA (98 bp; only one strand is listed): 5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17) Specifically, using a real-time PCR apparatus (Stratagene, Mx3005P), 94 ° C.-2 minutes in the presence of each primer 1 ⁇ M, natural base substrate dNTP 0.2 mM, artificial base substrate Cy3-hx-dPxTP 2 ⁇ M
  • the PCR reaction scale is 25 ⁇ l, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 ⁇ g / ml BSA, 1 ⁇ Titanium Taq DNA polymerase.
  • the DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration.
  • the filter set used for detection is excitation 545 nm-fluorescence 568 nm (for CY3).
  • attached analysis software MxPro version 4.10 was used.
  • Example 16 Detection of real-time PCR product on gel electrophoresis with quenching Px base bound to fluorescent molecule Cy3 (FIG. 23) Since the PCR product shown in FIG. 22 incorporates Cy3, when this product is electrophoresed on an agarose gel, the PCR product can be run on the gel without using a conventional DNA staining dye such as EtBr or SYBR Green. Detection is possible with Cy3 fluorescence. In FIG. 23, 12 ⁇ l of the PCR product shown in FIG.
  • Example 17 Fluorescence characteristics of DNA containing fluorescent molecule Cy3 and fluorescent artificial base S (FIG. 24) DNA fragments purified by HPLC after chemical synthesis were prepared with 10 mM sodium phosphate buffer (pH 7) containing 100 mM NaCl and 0.1 mM EDTA so that the final concentration would be 5 ⁇ M. The results of examining the spectrum are shown in FIG.
  • UV irradiation was performed from below using a UV transilluminator.
  • a DNA fragment containing one fluorescent artificial base s emits light when irradiated at 254 nm, 302 nm, and 365 nm (photo lane 2), and when two s are introduced into the DNA adjacent to each other, the fluorescence is quenched (photo lane). 3).
  • the DNA into which Cy3 was introduced emitted little light when irradiated with 254 nm and 302 nm, but hardly emitted when irradiated with 365 nm (Photo Lane 4).
  • FIG. 25 shows the principle of real-time PCR when PCR is performed in the presence of a Cy3-hx-dPxTP substrate using a primer containing two adjacent artificial bases Ds and fluorescent artificial bases s.
  • Ds is arranged in the vicinity of S and Cy3-hx is complemented and specifically incorporated in the Ds
  • FRET occurs between S and Cy3 by irradiation around 365 nm, and PCR amplified double-stranded DNA Only can be shined specifically.
  • FIG. 26 shows the result of visually confirming the product that was subjected to PCR 25 cycles using the following DNA fragment.
  • each primer 1 ⁇ M, natural base substrate dNTP each 0.2 mM, artificial base substrate Cy3-hx-dPxTP 2 ⁇ M In the presence of the sample, 25 cycles of PCR were performed under the conditions of 2-step PCR, with 94 ° C for 5 minutes and 68 ° C for 40 seconds after 94 ° C for 2 minutes.
  • the reaction scale of PCR is 25 ⁇ l, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 ⁇ g / ml BSA, 1 ⁇ Titanium Taq DNA polymerase.
  • the DNA fragment concentration used for the template is 0.5 nM.
  • SYBR Green I instead of the artificial base substrate Cy3-hx-dPxTP 2 ⁇ M, SYBR Green I (final concentration 1/30000) and ROX (final concentration 1/500) as a reference dye are used. Added.
  • the PCR reaction scale is 25 ⁇ l, and the composition of the reaction solution is 40 mM Tricine-KOH pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 ⁇ g / ml BSA, 1 ⁇ Titanium Taq DNA polymerase.
  • the DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration.
  • FIG. 27a shows the case where the visualized PCR product of FIG. 27a is electrophoresed on an agarose gel, and the product can be detected by FRET from s to Cy3 by 312 nm irradiation, and the fluorescence of Cy3 directly incorporated into DNA by 532 nm irradiation. It was confirmed that product detection was possible.
  • Sequence used for experiment (underlined part corresponds to primer annealing site) 5′-primer sequence: 5′- CATGTAGATGCCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15) 3′-primer sequence: 5′-AATAAssGCDs TCCTCAAAGGTGGTGACTTC- 3 ′ (SEQ ID NO: 26) Double-stranded template DNA (98 bp; only one strand is listed): 5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17) The PCR reaction was carried out using a real-time PCR apparatus (Stratagene, Mx3005P) at 94 ° C.
  • PCR was performed for 30, 35, 40, 45, and 55 cycles under conditions of 2-step PCR with 94 ° C. for 5 seconds and 68 ° C. for 40 seconds as one cycle.
  • the reaction scale of PCR is 25 ⁇ l, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 ⁇ g / ml BSA, 1 ⁇ Titanium Taq DNA polymerase.
  • the DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration.
  • Quantitative analysis by image processing in the tube after completion of the reaction was performed according to the following procedure.
  • the image was taken with a digital camera through a UV cut filter and an orange filter under UV irradiation of 365 nm from below, and the resulting file (JPEG format) was taken from Adobe Photoshop ver.
  • the image mode was converted to a Tiff format file with a gray scale and a resolution of 72 pixels / inch. This file was read with Science Lab 2005 Multi Gauge software for quantitative analysis.
  • Example 20 PCR product detection method using a nucleoside derivative (s-hx-dU) in which a fluorescent molecule (s base) is bound to a natural base via a linker and a Ds-Px base pair (FIGS. 29b to 29d) )
  • This example is a counter example of FIG. 29a.
  • PCR was performed in the presence of a Cy3-hx-dPxTP substrate using a primer containing two adjacent modified bases (s-hx-dU) in which s, which is a fluorescent artificial base, was bound to a natural base U via a linker.
  • s-hx-dU primer containing two adjacent modified bases
  • FIG. 29a The principle of real-time PCR when performing is shown in FIG. 29a. When two s-hx-dUs are introduced adjacently, the fluorescence of s is quenched as in the case where two s are introduced adjacently (FIG. 25).
  • a site for color development containing an artificial base can be introduced into any part of the primer and can be used for PCR methods such as the LAMP method and the SMAP method.
  • this technique can be used in addition to primer regions such as padlock PCR.
  • FIG. 29b shows the DNA sequence used and the PCR conditions.
  • FIG. 29c shows the results of 55 cycles of real-time PCR, and
  • FIG. 29d shows the results of visual confirmation of amplification products after 55 cycles of PCR.
  • PCR amplification was performed between 0 and 3000000 copies of target DNA (target DNA)
  • visual determination was possible with 3 or more copies.
  • the reaction scale of PCR is 25 ⁇ l, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4, 3.75 ⁇ g / ml BSA, 1 ⁇ Titanium Taq DNA polymerase.
  • the DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration.
  • the reaction tube was directly irradiated with 365 nm UV, and fluorescence was detected visually through an orange filter.
  • FIG. 30 Synthesis of 8-bromo-1-octyne (step (a) in FIG. 30)
  • Dehydrated dichloromethane (20 ml) and triphenylphosphine (5.91 g, 22.5 mmol) were added to 8-hydroxy-1-octyne (1.95 g, 15 mmol), cooled to 0 ° C., and dissolved in dehydrated dichloromethane (10 ml).
  • Carbon tetrabromide (7.46 g, 22.5 mmol) was added dropwise and stirred at room temperature for 2 hours.
  • 6- (thien-2-yl) -9- (7-octynyl) -2-aminopurine (1.60 g, 4.91 mmol) obtained in 2) was added and stirred at room temperature overnight.
  • dehydrated tetrahydrofuran (4.5 ml), dehydrated diisopropylethylamine (235 ⁇ l, 1.35 mmol) and 2-cyanoethyl N, N′-diisopropylchlorophosphoramidide (241 ⁇ l, 1.08 mmol) were added and stirred at room temperature for 1 hour. did.
  • Example 22 Chemical synthesis of Dss-hx-dU amidite reagent (compound of FIG. 6) (FIG. 33) 1) Synthesis of 7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine (step (a) in FIG. 33) A solution of 7- (2,2′-bitien-5-yl) -imidazo [4,5-b] pyridine (850 mg, 3.0 mmol) and potassium carbonate (1.3 g, 9.0 mmol) in DMF (15 ml) was added. Stir at 60 ° C. for 1 hour.

Abstract

Disclosed is a nucleic acid base analog with quenching characteristics and fluorescence and application thereof. Specifically disclosed is a quencher characterized by having a 2-nitropyrrole structure shown by formula (I). [In formula (I), R1 and R2 are groups selected independently from a group formed from ribose and deoxyribose; hydrogen, hydroxyl groups, SH groups, and halogens; optionally substituted C2-10 alkyl groups, alkenyl groups, and alkynyl groups; one or a plurality of 5-member heterocycles, one or a plurality of 6-member heterocycles, one or a plurality of polycyclic heterocycles and one or a plurality of aromatic rings, containing nitrogen atoms or sulfur atoms; sugars, sugar chains, amino acids, and peptides; and florescent molecules bound through linkers.]

Description

消光性ならびに蛍光性の核酸塩基類似体とその応用Quenching and fluorescent nucleobase analogues and their applications
　本出願は、２０１０年４月２１日に提出された日本国出願特願２０１０−０９８３１９に基づく優先権を主張し、その全内容は本明細書中に取り込まれる。
　本発明は、消光性ならびに蛍光性の核酸塩基類似体とその応用に関する。
　具体的には、本発明は、２−ニトロピロールとその１位と４位修飾体、ならびにそれらのヌクレオシド誘導体が消光性の分子あるいは核酸塩基類似体として機能することの発見とその利用に関するものであり、ＰＣＲ産物の可視化などの幅広い検出・診断に用いることができる。 This application claims priority based on Japanese Patent Application No. 2010-098319 filed on Apr. 21, 2010, the entire contents of which are incorporated herein.
The present invention relates to quenching and fluorescent nucleobase analogs and their applications.
Specifically, the present invention relates to the discovery and utilization of 2-nitropyrrole and its modified 1- and 4-positions, and their nucleoside derivatives as quenching molecules or nucleobase analogs. Yes, it can be used for a wide range of detection and diagnosis such as visualization of PCR products.
　人工的に新たな塩基対を作り出しＤＮＡの遺伝情報を拡張する技術は、汎用性の高い２つの応用が考えられ、人工塩基対の開発研究が盛んに行われている。応用の１つは、複製・転写・翻訳で機能する人工塩基対を作り出し、新たな構成成分を組み込んだＤＮＡ、ＲＮＡ、タンパク質を作り出すことである。もう１つの応用は、人工塩基対をＤＮＡやＲＮＡに組み込んだ二本鎖核酸を形成させることで、核酸断片のプローブの配列種を増やすことによりリアルタイムＰＣＲによるマルチプレックスやＤＮＡコンピュータへの応用、さらに翻訳でタンパク質中に人工のアミノ酸を導入する際の新たなコドンとアンチコドンに用いることができる。 The technology for artificially creating new base pairs and extending DNA genetic information is considered to have two highly versatile applications, and research and development of artificial base pairs has been actively conducted. One application is to create artificial base pairs that function in replication, transcription, and translation to create DNA, RNA, and proteins that incorporate new components. Another application is the formation of double-stranded nucleic acids in which artificial base pairs are incorporated into DNA or RNA, thereby increasing the number of nucleic acid fragment probe sequence species to apply to real-time PCR multiplexes and DNA computers. It can be used as a new codon and anticodon when introducing artificial amino acids into proteins by translation.
　蛍光性を示す核酸塩基類似体は数多く報告されているが、核酸塩基の類似体自身が強い消光作用を示す物質はこれまで知られていない。従来、核酸塩基にリンカーを介してダブシル基などの消光性分子を結合させる方法が知られている。しかしながら、この場合には塩基対を形成する塩基が、近傍に位置する蛍光性分子とは完全に接していないので、消光作用が弱くなり、その検出は装置に頼らないと不可能であった。本発明前は、塩基の消光作用を利用した塩基対の簡便かつ効率的な検出方法はみいだされていなかった。 Numerous nucleobase analogues exhibiting fluorescence have been reported, but no substance has been known so far that the nucleobase analogue itself exhibits a strong quenching action. Conventionally, a method of binding a quenching molecule such as a dabsyl group to a nucleobase via a linker is known. However, in this case, the base forming the base pair is not completely in contact with the fluorescent molecule located in the vicinity, so that the quenching action is weakened, and its detection is impossible without relying on an apparatus. Prior to the present invention, a simple and efficient detection method for base pairs utilizing the quenching action of bases has not been found.
ＷＯ２００９／１２３２１６WO2009 / 123216 特願２００９−２３２７７６（２００９年１０月６日出願）Japanese Patent Application No. 2009-232737 (filed on Oct. 6, 2009) 特開２００７−０６１０８７JP2007-061087 特願２００９−２３２８５１（２００９年１０月６日出願）Japanese Patent Application No. 2009-232851 (filed on October 6, 2009)
　本発明者らは、消光性を示す塩基を見いだし、そして、蛍光性を示す塩基と消光性を示す塩基とが選択的に塩基対を形成すれば、ＤＮＡが二本鎖を形成すると蛍光性人工塩基を強く消光させることができる可能性があり、ＰＣＲにおけるＤＮＡ増幅やモレキュラービーコンなどで目視判定が可能な検出技術を作り出すことができると考え、本発明に想到した。 The present inventors have found a base exhibiting quenching properties, and if a base exhibiting fluorescence and a base exhibiting quenching properties selectively form a base pair, when DNA forms a double strand, a fluorescent artificial base is formed. The present inventors have conceived that the present invention has been conceived because it is possible that a base can be strongly quenched, and that a detection technique that can be visually judged by DNA amplification or molecular beacons in PCR can be created.
　２−ニトロピロール誘導体は、本発明者らがこれまで開発してきた人工塩基対のＰｎやＰｘで表される塩基である。ＰｎやＰｘは、第三の核酸塩基対（人工塩基対）として、その相補人工塩基（Ｄｓ：７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基）と塩基対（Ｄｓ−Ｐｎ、Ｄｓ−Ｐｘ塩基対）を形成し、複製や転写で核酸中の特定部位に導入できることが示されている。また、蛍光性の人工塩基としてＤｓの改良塩基であるＤｓｓ（７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン）とも２−ニトロピロールが塩基対を形成する。 The 2-nitropyrrole derivative is a base represented by Pn or Px, which is an artificial base pair that the present inventors have developed so far. Pn and Px are used as a third nucleic acid base pair (artificial base pair) as a complementary artificial base (Ds: 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group) and a base pair ( Ds-Pn, Ds-Px base pairs), and it is shown that they can be introduced into specific sites in nucleic acids by replication or transcription. In addition, 2-nitropyrrole forms a base pair with Dss (7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridine), which is an improved base of Ds, as a fluorescent artificial base. To do.
　本発明者らは本発明において、２−ニトロピロールに消光性があることを初めて明らかにした。例えば、ＰｎやＰｘが二本鎖ＤＮＡ中でＤｓｓと塩基対を形成すると２−ニトロピロールの消光性によりＤｓｓの蛍光が消光されることを見いだした。なお、２−ニトロピロールに類似の３−ニトロピロールは、ユニバーサル塩基として知られているが、本発明の２−ニトロピロールと異なり消光作用は低い。 The inventors of the present invention revealed for the first time that 2-nitropyrrole has a quenching property in the present invention. For example, it was found that when Pn or Px forms a base pair with Dss in double-stranded DNA, the fluorescence of Dss is quenched by the quenching property of 2-nitropyrrole. In addition, although 3-nitropyrrole similar to 2-nitropyrrole is known as a universal base, the quenching action is low unlike 2-nitropyrrole of the present invention.
　ＤｓｓとＰｎ（あるいはＰｘ）の塩基対を含む二本鎖核酸は、蛍光性のＤｓｓに消光性のＰｎ（あるいはＰｘ）が塩基対として接しているので、Ｄｓｓの蛍光は効率よく消光される。しかし、二本鎖構造が一本鎖のＤＮＡに変性した場合は、Ｄｓｓを含むＤＮＡ鎖が発光する。このような人工塩基対はこれまでに例が無く、この性質を利用して、新たなモレキュラービーコンなどの検出・診断技術が可能になった。 In the double-stranded nucleic acid containing the base pair of Dss and Pn (or Px), the quenching Pn (or Px) is in contact with the fluorescent Dss as a base pair, so that the fluorescence of Dss is efficiently quenched. However, when the double-stranded structure is denatured into single-stranded DNA, a DNA strand containing Dss emits light. Such an artificial base pair has never been seen before, and by using this property, a new molecular beacon detection / diagnosis technique has become possible.
　２−ニトロピロールの４位にリンカーを介して蛍光色素を結合させると、そのヌクレオシドやヌクレオチド誘導体（Ｐｘの誘導体）は２−ニトロピロールと色素が相互作用することによりその蛍光がある程度消光されることが分かった。しかしこのヌクレオチド誘導体がＤＮＡやＲＮＡ中に導入されると、２−ニトロピロールと相互作用していた色素部分がＤＮＡやＲＮＡ断片中の外側に飛び出すので、本来の蛍光強度が復活することも見出した。また、このＰｘの誘導体の基質（ヌクレオシド三リン酸）は、鋳型中の人工塩基Ｄｓに相補して、複製によりＤＮＡ中に導入できる。これらのＰｘ誘導体の蛍光変化の特性と複製におけるＤＮＡ中への部位特異的な取り込みを利用することにより、リアルタイムＰＣＲなどの検出・診断技術に本技術を利用することができる。 When a fluorescent dye is attached to the 4-position of 2-nitropyrrole via a linker, the fluorescence of the nucleoside or nucleotide derivative (Px derivative) is quenched to some extent by the interaction of 2-nitropyrrole and the dye. I understood. However, when this nucleotide derivative was introduced into DNA or RNA, the dye portion that interacted with 2-nitropyrrole jumped out of the DNA or RNA fragment, and the original fluorescence intensity was restored. . Moreover, the substrate (nucleoside triphosphate) of the derivative of Px can be introduced into DNA by duplication, complementing the artificial base Ds in the template. By utilizing the characteristics of fluorescence change of these Px derivatives and site-specific incorporation into DNA in replication, the present technology can be used for detection / diagnosis techniques such as real-time PCR.
　また、発明者らが別途開発した蛍光性人工塩基（ｓ）と本Ｄｓ−Ｐｘ塩基対を組み合わせることにより、ＤＮＡのＰＣＲ増幅の可視化法を新たに開発した。この可視化ＰＣＲは、ＤＮＡ増幅を裸眼で識別できるため、従来のリアルタイムＰＣＲでは不可能であった臨床現場での迅速・簡便なＰＣＲ診断法として用いることができ、テーラーメイド医療を目指したコンパニオン診断薬としての道を開く。また、本技術による特定のＤＮＡ配列の検出は、医療に限らず、ビールなどの発酵食品の品質管理（酵母の遺伝子変異を検出）や輸入食材の流通管理（食品の遺伝子による真贋判定）などにも応用できる。 In addition, by combining the fluorescent artificial base (s) separately developed by the inventors and the present Ds-Px base pair, a method for visualizing PCR amplification of DNA was newly developed. Since this visualization PCR can identify DNA amplification with the naked eye, it can be used as a rapid and simple PCR diagnostic method in clinical settings, which was impossible with conventional real-time PCR, and as a companion diagnostic agent aimed at tailor-made medicine Open the way. In addition, the detection of specific DNA sequences by this technology is not limited to medical treatment, but for quality control of fermented foods such as beer (detecting genetic mutations in yeast) and distribution management of imported foods (determination of authenticity based on food genes). Can also be applied.
　以上より、限定されるわけではないが、本発明は以下の態様を含む。
［態様１］
　式Ｉで示す２−ニトロピロール構造を有する消光剤 As described above, the present invention includes the following aspects, although not limited thereto.
[Aspect 1]
Quenching agent having 2-nitropyrrole structure represented by formula I
Figure JPOXMLDOC01-appb-C000021
Figure JPOXMLDOC01-appb-C000021
　［式Ｉにおいて、Ｒ１及びＲ２は
　リボース、デオキシリボース、
　水素、水酸基、ＳＨ基、ハロゲン、
　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
　糖、糖鎖、アミノ酸、ペプチド、
　リンカーを介して結合した蛍光性分子
からなる群から独立に選択される基である］。
［態様２］
　式Ｉにおいて、Ｒ１がリボースまたはデオキシリボースである、態様１に記載の消光剤。
［態様３］
　人工塩基対の形成を検出する方法であって、
１）式ＩＩ [In Formula I, R 1 and R 2 are ribose, deoxyribose,
Hydrogen, hydroxyl group, SH group, halogen,
A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
Sugar, sugar chain, amino acid, peptide,
A group independently selected from the group consisting of fluorescent molecules linked via a linker.
[Aspect 2]
The quencher according to embodiment 1, wherein in formula I, R 1 is ribose or deoxyribose.
[Aspect 3]
A method for detecting the formation of an artificial base pair,
1) Formula II
Figure JPOXMLDOC01-appb-C000022
Figure JPOXMLDOC01-appb-C000022
　［式ＩＩにおいて、Ｒ２は、
　水素、水酸基、ＳＨ基、ハロゲン、
　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
　糖、糖鎖、アミノ酸、ペプチド、
　リンカーを介して結合した蛍光性分子、
からなる群から選択される基である］
で表される消光性人工塩基を有するヌクレオシドまたはヌクレオチド、あるいは、
２）蛍光共鳴エネルギー転移（ＦＲＥＴ）や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するヌクレオシドまたはヌクレオチド
のいずれか、あるいは双方を用いることを特徴とする、前記方法。
［態様４］
　人工塩基の塩基対の形成を検出する方法であって、蛍光性人工塩基と式ＩＩ [In Formula II, R 2 is
Hydrogen, hydroxyl group, SH group, halogen,
A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
Sugar, sugar chain, amino acid, peptide,
A fluorescent molecule bound via a linker,
A group selected from the group consisting of]
A nucleoside or nucleotide having a quenching artificial base represented by:
2) Either a natural base modified with self-quenching, an artificial base, or a nucleoside or nucleotide having a base analog that can be a donor such as fluorescence resonance energy transfer (FRET) or static quenching, Alternatively, both methods are used.
[Aspect 4]
A method for detecting the formation of a base pair of an artificial base comprising a fluorescent artificial base and formula II
Figure JPOXMLDOC01-appb-C000023
Figure JPOXMLDOC01-appb-C000023
　［式ＩＩにおいて、Ｒ２は、
　水素、水酸基、ＳＨ基、ハロゲン、
　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
　糖、糖鎖、アミノ酸、ペプチド、
　リンカーを介して結合した蛍光性分子、
からなる群から選択される基である］
で表される消光性人工塩基との塩基対形成により、蛍光性人工塩基の蛍光の低下を観察することにより、人工塩基対が形成されたことが検出される、前記方法。
［態様５］
　蛍光性人工塩基の蛍光の低下により、人工塩基の塩基対の形成を検出する方法であって、以下の、
　（ｉ）７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）；
　（ｉｉ）７−（２，２’，５’，２’’−ターチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓｓ）；
　（ｉｉｉ）２−アミノ−６−（２，２’−ビチエン−５−イル）プリン−９−イル基（ｓｓ）；
　（ｉｖ）２−アミノ−６−（２，２’，５’，２’’−ターチエン−５−イル）プリン−９−イル基（ｓｓｓ）；
　（ｖ）４−（２，２’−ビチエン−５−イル）−ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｓ）；
　（ｖｉ）４−［２−（２−チアゾリル）チエン−５−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｖ）；及び
　（ｖｉｉ）４−［５−（２−チエニル）チアゾール−２−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｖａｓ）；
からなる群より選択される蛍光性人工塩基と、
　以下の式ＩＩＩ，又は式ＩＶの消光性塩基 [In Formula II, R 2 is
Hydrogen, hydroxyl group, SH group, halogen,
A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
Sugar, sugar chain, amino acid, peptide,
A fluorescent molecule bound via a linker,
A group selected from the group consisting of]
The method as described above, wherein the formation of an artificial base pair is detected by observing a decrease in fluorescence of the fluorescent artificial base by forming a base pair with the quenching artificial base represented by formula (1).
[Aspect 5]
A method for detecting the formation of a base pair of an artificial base by lowering the fluorescence of a fluorescent artificial base, comprising:
(I) 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss);
(Ii) 7- (2,2 ′, 5 ′, 2 ″ -tertien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dsss);
(Iii) 2-amino-6- (2,2′-bithien-5-yl) purin-9-yl group (ss);
(Iv) 2-amino-6- (2,2 ′, 5 ′, 2 ″ -tert-en-5-yl) purin-9-yl group (sss);
(V) 4- (2,2′-bithien-5-yl) -pyrrolo [2,3-b] pyridin-1-yl group (Dsas);
(Vi) 4- [2- (2-thiazolyl) thien-5-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dsav); and (vii) 4- [5- (2-thienyl) ) Thiazol-2-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dvas);
A fluorescent artificial base selected from the group consisting of:
Quenching bases of the following formula III or formula IV
Figure JPOXMLDOC01-appb-C000024
Figure JPOXMLDOC01-appb-C000024
　［式ＩＩＩにおいて、Ｒ３は、
　−Ｈ、ヨード、−ＣＨ３、 [In Formula III, R 3 is
-H, iodine, -CH 3,
Figure JPOXMLDOC01-appb-C000025
Figure JPOXMLDOC01-appb-C000025
　から選択される］ Selected from]
Figure JPOXMLDOC01-appb-C000026
Figure JPOXMLDOC01-appb-C000026
　［式ＩＶにおいて、Ｒ４は、
　−ＣＨ３、−ＣＨ２−ＮＨ２、及び [In Formula IV, R 4 is
-CH 3, -CH 2 -NH 2 and,
Figure JPOXMLDOC01-appb-C000027
Figure JPOXMLDOC01-appb-C000027
　（ここにおいて、ｎは０ないし１２のいずれかの整数である）
から選択される］
との間で塩基対が形成されると、蛍光性人工塩基の蛍光が低下し、人工塩基対が形成されたことが検出される、前記方法。
［態様６］
　７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）を塩基として有するポリヌクレオチドを含む、核酸プライマー；並びに、
　以下の式ＩＩＩ、又は式ＩＶの消光性塩基 (Where n is an integer from 0 to 12)
Selected from]
When the base pair is formed between the fluorescent artificial base, the fluorescence of the fluorescent artificial base decreases, and it is detected that the artificial base pair is formed.
[Aspect 6]
A nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base; and
A quenching base of the following formula III or formula IV
Figure JPOXMLDOC01-appb-C000028
Figure JPOXMLDOC01-appb-C000028
　［式ＩＩＩにおいて、Ｒ３は、
　−Ｈ、ヨード、−ＣＨ３、 [In Formula III, R 3 is
-H, iodine, -CH 3,
Figure JPOXMLDOC01-appb-C000029
 から選択される］
Figure JPOXMLDOC01-appb-C000029
 Selected from]
Figure JPOXMLDOC01-appb-C000030
Figure JPOXMLDOC01-appb-C000030
　［式ＩＶにおいて、Ｒ４は、
　−ＣＨ３、−ＣＨ２−ＮＨ２、及び [In Formula IV, R 4 is
-CH 3, -CH 2 -NH 2 and,
Figure JPOXMLDOC01-appb-C000031
Figure JPOXMLDOC01-appb-C000031
　（ここにおいて、ｎは０ないし１２のいずれかの整数である）
から選択される］
を塩基として有するポリヌクレオチド
を含む、蛍光性人工塩基の蛍光の低下により、人工塩基の塩基対の形成を検出する方法に使用するためのキット。
［態様７］
　人工塩基対を検出する方法であって、
　式Ｖ (Where n is an integer from 0 to 12)
Selected from]
A kit for use in a method for detecting the formation of a base pair of an artificial base by reducing the fluorescence of the fluorescent artificial base, comprising a polynucleotide having a base as a base.
[Aspect 7]
A method for detecting artificial base pairs,
Formula V
Figure JPOXMLDOC01-appb-C000032
Figure JPOXMLDOC01-appb-C000032
　［式Ｖにおいて、Ｒ５は、リンカーを介して結合した蛍光性分子である］
で表される消光性人工塩基中の蛍光性分子の蛍光強度が、式Ｖの人工塩基が塩基対を形成することによって変化し、人工塩基対が形成されたことが検出される、前記方法。
［態様８］
　蛍光強度の変化により、人工塩基の塩基対の形成を検出する方法であって、
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、以下の式ＶＩの塩基： [In Formula V, R 5 is a fluorescent molecule bound via a linker]
The method, wherein the fluorescence intensity of the fluorescent molecule in the quenching artificial base represented by formula (1) is changed by the formation of the base pair by the artificial base of Formula V, and the formation of the artificial base pair is detected.
[Aspect 8]
A method for detecting the formation of a base pair of an artificial base by a change in fluorescence intensity,
7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
Figure JPOXMLDOC01-appb-C000033
Figure JPOXMLDOC01-appb-C000033
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
　式ＶＩの塩基中の蛍光性分子の蛍光強度が増加し、人工塩基対が形成されたことが検出される、前記方法。
［態様９］
　蛍光性分子が、インドカルボシアニン（Ｃｙ３）、インドジカルボシアニン（Ｃｙ５）、５−カルボキシフルオレセイン（５−ＦＡＭ）、６−カルボキシフルオレセイン（６−ＦＡＭ）、５−カルボキシテトラメチルローダミン（５−ＴＡＭＲＡ）、６−カルボキシテトラメチルローダミン（６−ＴＡＭＲＡ）、５−ジメチルアミノナフタレン−１−スルホン酸（ＤＡＮＳＹＬ）、５−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（５−ＨＥＸ）、６−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（６−ＨＥＸ）、５−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（５−ＴＥＴ）、６−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（６−ＴＥＴ）、５−カルボキシ−Ｘ−ローダミン（５−ＲＯＸ）、及び６−カルボキシ−Ｘ−ローダミン（６−ＲＯＸ）からなる群から選択される、態様７又は８に記載の方法。
［態様１０］
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）を塩基として有するポリヌクレオシドを含む、核酸プライマー、並びに、
　式ＶＩの塩基： In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
Said method wherein the fluorescence intensity of the fluorescent molecule in the base of formula VI is increased and it is detected that an artificial base pair has been formed.
[Aspect 9]
Fluorescent molecules include indocarbocyanine (Cy3), indodicarbocyanine (Cy5), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA) ), 6-carboxytetramethylrhodamine (6-TAMRA), 5-dimethylaminonaphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (5-HEX), 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (6-HEX), 5-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (5-TET), 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein (6-TET), 5- Rubokishi -X- rhodamine (5-ROX), and is selected from the group consisting of 6-carboxy -X- rhodamine (6-ROX), method according to embodiment 7 or 8.
[Aspect 10]
A nucleic acid primer comprising a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base; and
Base of formula VI:
Figure JPOXMLDOC01-appb-C000034
Figure JPOXMLDOC01-appb-C000034
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］を塩基として有するポリヌクレオチド
を含む、蛍光強度の変化により、人工塩基の塩基対の形成を検出する方法に使用するためのキット。
［態様１１］
　人工塩基対の形成を検出する方法であって、
　蛍光共鳴エネルギー転移（ＦＲＥＴ）や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドを含む核酸を利用し、当該核酸中の人工塩基（第一人工塩基）と蛍光性分子を有する人工塩基（第二人工塩基）との間で人工塩基対が形成されると、前記天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドから、第二人工塩基の有する蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）により、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、前記方法。
［態様１２］
　蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
　７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）と、以下の式ＶＩの塩基： [In Formula VI, R 6 is a fluorescent molecule bound through or without a linker], and detects the formation of an artificial base base pair by a change in fluorescence intensity. Kit for use in the method.
[Aspect 11]
A method for detecting the formation of an artificial base pair,
Using a nucleic acid containing a polynucleoside having a natural base modifier, an artificial base, or a base analog having a self-quenching property that can serve as a donor such as fluorescence resonance energy transfer (FRET) and static quenching (static quenching), When an artificial base pair is formed between an artificial base (first artificial base) in the nucleic acid and an artificial base having a fluorescent molecule (second artificial base), the modified natural base, artificial base, or base Detects the formation of an artificial base pair by changing the fluorescence spectrum due to fluorescence resonance energy transfer or static quenching from a polynucleoside having an analog to a fluorescent molecule of a second artificial base Said method.
[Aspect 12]
A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum due to fluorescence resonance energy transfer or static quenching,
7- (2,2′-Bitien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) and a base of formula VI below:
Figure JPOXMLDOC01-appb-C000035
Figure JPOXMLDOC01-appb-C000035
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
　２４０−４１０ｎｍの紫外線による励起により、Ｄｓｓから式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などが生じて、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、前記方法。
［態様１３］
　蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、以下の式ＶＩの塩基： In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
Excitation with 240-410 nm ultraviolet light causes fluorescence resonance energy transfer, static quenching, etc. from Dss to the fluorescent molecule in the base of formula VI, and the fluorescence spectrum changes, artificial base pairs become The method, wherein the formation is detected.
[Aspect 13]
A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum due to fluorescence resonance energy transfer or static quenching,
7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
Figure JPOXMLDOC01-appb-C000036
Figure JPOXMLDOC01-appb-C000036
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
　２４０−３９０ｎｍの紫外線による励起により、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などにより、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）を塩基として有するポリヌクレオチドが少なくとも１つ存在する、前記方法。
［態様１４］
　蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓ）と、以下の式ＶＩの塩基： In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI by excitation with ultraviolet light at 240-390 nm It is detected that the fluorescence spectrum is changed due to static quenching or the like, and an artificial base pair is formed.
Here, at least one polynucleotide having a 2-amino-6- (2-thienyl) purin-9-yl group (s) as a base is present on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. Said method.
[Aspect 14]
A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum due to fluorescence resonance energy transfer or static quenching,
7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
Figure JPOXMLDOC01-appb-C000037
Figure JPOXMLDOC01-appb-C000037
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
　３５０−３９０ｎｍの紫外線による励起により、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などにより蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドが少なくとも１つ存在する、
前記方法。
［態様１５］
　蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、以下の式ＶＩの塩基： In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI upon excitation with UV light at 350-390 nm The fluorescence spectrum is changed by static quenching or the like, and it is detected that an artificial base pair is formed.
Here, a base in which at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) is bound to a natural base on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. There is at least one polynucleotide having
Said method.
[Aspect 15]
A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum due to fluorescence resonance energy transfer or static quenching,
7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
Figure JPOXMLDOC01-appb-C000038
Figure JPOXMLDOC01-appb-C000038
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
　２４０−４１０ｎｍの紫外線による励起により、７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などにより蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、少なくとも１つの７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドが存在する、
前記方法。
［態様１６］
　蛍光性物質が、インドカルボシアニン（Ｃｙ３）、インドジカルボシアニン（Ｃｙ５）、５−カルボキシフルオレセイン（５−ＦＡＭ）、６−カルボキシフルオレセイン（６−ＦＡＭ）、５−カルボキシテトラメチルローダミン（５−ＴＡＭＲＡ）、６−カルボキシテトラメチルローダミン（６−ＴＡＭＲＡ）、５−ジメチルアミノナフタレン−１−スルホン酸（ＤＡＮＳＹＬ）、５−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（５−ＨＥＸ）、６−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（６−ＨＥＸ）、５−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（５−ＴＥＴ）、６−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（６−ＴＥＴ）、５−カルボキシ−Ｘ−ローダミン（５−ＲＯＸ）、及び６−カルボキシ−Ｘ−ローダミン（６−ＲＯＸ）からなる群から選択される、態様１１ないし１５のいずれか１項に記載の方法。
［態様１７］
　式ＶＩの塩基中の置換基Ｒ６が以下の： In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
Fluorescence in the base of formula VI from 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) upon excitation with 240-410 nm UV light The fluorescence spectrum is changed by fluorescence resonance energy transfer to the molecule or static quenching, and it is detected that an artificial base pair is formed.
Here, at least one 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group on the same strand as the nucleic acid containing a polynucleoside having Ds as a base There is a polynucleotide having a base in which (Dss) is bound to a natural base,
Said method.
[Aspect 16]
Fluorescent substances include indocarbocyanine (Cy3), indodicarbocyanine (Cy5), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA) ), 6-carboxytetramethylrhodamine (6-TAMRA), 5-dimethylaminonaphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (5-HEX), 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (6-HEX), 5-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (5-TET), 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein (6-TET), 5- Rubokishi -X- rhodamine (5-ROX), and is selected from the group consisting of 6-carboxy -X- rhodamine (6-ROX), The method according to any one of embodiments 11 to 15.
[Aspect 17]
The substituent R 6 in the base of formula VI is:
Figure JPOXMLDOC01-appb-C000039
Figure JPOXMLDOC01-appb-C000039
　の構造を有する、態様１２−１５に記載の方法。
［態様１８］
　検出スペクトルの変化が、肉眼で判定できる、態様１１ないし１７のいずれか１項に記載の方法。
［態様１９］
　核酸の塩基対が、転写、逆転写、複製又は翻訳の工程で形成される、態様１１ないし１８のいずれか１項に記載の方法。
［態様２０］
　以下のｉ）−ｉｉｉ）からなる群から選択される、１つの核酸プライマー；
　ｉ）７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）を塩基として有するポリヌクレオチドを含む、核酸プライマー；
　ｉｉ）７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）を塩基として有するポリヌクレオシド、及び、少なくとも１つの、２−アミノ−６−（２−チエニル）−９Ｈ−プリン−９−イル基（ｓ）を塩基として有するポリヌクレオチドを含む、核酸プライマー；
　ｉｉｉ）７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓ）を塩基として有するポリヌクレオシド、及び、少なくとも１つの、２−アミノ−６−（２−チエニル）−９Ｈ−プリン−９−イル基（ｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドを含む、核酸プライマー；及び
　ｉｖ）７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）を塩基として有するポリヌクレオシド、及び、７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドを含む、核酸プライマー、
　並びに、式ＶＩの塩基： The method according to embodiments 12-15, having the structure:
[Aspect 18]
The method according to any one of aspects 11 to 17, wherein the change in the detection spectrum can be determined with the naked eye.
[Aspect 19]
The method according to any one of aspects 11 to 18, wherein the nucleic acid base pair is formed by a transcription, reverse transcription, replication or translation process.
[Aspect 20]
One nucleic acid primer selected from the group consisting of i) -iii) below:
i) a nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base;
ii) a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base, and at least one 2-amino-6- (2-thienyl)- A nucleic acid primer comprising a polynucleotide having a 9H-purin-9-yl group (s) as a base;
iii) a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base, and at least one 2-amino-6- (2-thienyl) A nucleic acid primer comprising a polynucleotide having a base to which a -9H-purin-9-yl group (s) is bound to a natural base; and iv) 7- (2-thienyl) imidazo [4,5-b] pyridine 3 -A polynucleoside having an yl group (Ds) as a base and a 7- (2,2'-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) is a natural base A nucleic acid primer comprising a polynucleotide having a base bound to
And a base of formula VI:
Figure JPOXMLDOC01-appb-C000040
Figure JPOXMLDOC01-appb-C000040
　［式ＶＩにおいてここで、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］
を塩基として有するポリヌクレオチド
を含む、蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法に使用するためのキット。 [Wherein R 6 is a fluorescent molecule attached via or without a linker in Formula VI]
A kit for use in a method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum by fluorescence resonance energy transfer, static quenching, or the like, comprising a polynucleotide having a base as a base.
　Ｉ．消光剤
１．　消光剤の構造
　本発明は、新規な消光剤を提供する。本発明の消光剤は、式Ｉで示す２−ニトロピロール構造を有する I. Quencher 1. Quencher Structure The present invention provides a novel quencher. The quencher of the present invention has a 2-nitropyrrole structure represented by Formula I
Figure JPOXMLDOC01-appb-C000041
Figure JPOXMLDOC01-appb-C000041
　［式Ｉにおいて、Ｒ１及びＲ２は
　リボース、デオキシリボース、
　水素、水酸基、ＳＨ基、ハロゲン、
　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
　糖、糖鎖、アミノ酸、ペプチド、
　リンカーを介して結合した蛍光性分子
からなる群から独立に選択される基である］
ことを特徴とする。 [In Formula I, R 1 and R 2 are ribose, deoxyribose,
Hydrogen, hydroxyl group, SH group, halogen,
A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
Sugar, sugar chain, amino acid, peptide,
It is a group independently selected from the group consisting of fluorescent molecules linked via a linker.
It is characterized by that.
　本発明は、２−ニトロピロール構造が消光効果を有する、という発見に基づく。よって、Ｒ１及びＲ２は特に限定されず、任意の基を選択することが可能である。Ｒ１及びＲ２は任意に独立に選択される。 The present invention is based on the discovery that 2-nitropyrrole structures have a quenching effect. Therefore, R 1 and R 2 are not particularly limited, and any group can be selected. R 1 and R 2 are arbitrarily selected independently.
　ｉ）リボース、デオキシリボース
　Ｒ１及び／又はＲ２は、好ましくは、リボース、デオキシリボースである。好ましくは、Ｒ１はリボース、デオキシリボースである。 i) Ribose, deoxyribose R 1 and / or R 2 are preferably ribose or deoxyribose. Preferably, R 1 is ribose or deoxyribose.
　「リボース」とは、五炭糖の単糖の１種で、ＩＵＰＡＣ名は「（３Ｒ，４Ｓ，５Ｒ）−５−（ヒドロキシメチル）テトラヒドロフラン−２，３，４−トリオール」である。
　「デオキシリボース」とは、アルデヒド基を含む五炭糖の単糖の１種で、ＩＵＰＡＣ名は「（２Ｒ，４Ｓ，５Ｒ）−５−（ヒドロキシメチル）テトラヒドロフラン−２，４−ジオール」である。 “Ribose” is one of pentose monosaccharides, and the IUPAC name is “(3R, 4S, 5R) -5- (hydroxymethyl) tetrahydrofuran-2,3,4-triol”.
“Deoxyribose” is a kind of pentose monosaccharide containing an aldehyde group, and the IUPAC name is “(2R, 4S, 5R) -5- (hydroxymethyl) tetrahydrofuran-2,4-diol”. .
　本発明の消光剤は、好ましくは、ポリヌクレオシド又はポリヌクレオチド中の塩基として、人工塩基対を形成した場合に、塩基対を形成する相手方、あるいは、近傍に存在する塩基が蛍光性の塩基である場合に、その蛍光を消す消光効果を奏する。あるいは、塩基対を形成する相補性の人工塩基、あるいは、近傍に存在する塩基に結合した蛍光物質の蛍光を消光する。 In the quencher of the present invention, preferably, when an artificial base pair is formed as a base in a polynucleoside or polynucleotide, the partner that forms the base pair or a base that exists in the vicinity is a fluorescent base In some cases, it has a quenching effect that extinguishes the fluorescence. Or the fluorescence of the fluorescent substance couple | bonded with the complementary artificial base which forms a base pair, or the base which exists in the vicinity is quenched.
　ｉｉ）水素、水酸基、ＳＨ基、ハロゲン
　ハロゲンの種類は特に限定されない。好ましくは、フッ素、臭素、ヨウ素等からなる群から選択される。 ii) Hydrogen, hydroxyl group, SH group, halogen The type of halogen is not particularly limited. Preferably, it is selected from the group consisting of fluorine, bromine, iodine and the like.
　ｉｉｉ）置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基
　炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基は、直鎖状でも枝分かれしていてもよく、特に限定されない。好ましくは、メチル基、エチル基、プロピニル基、エチレン基、　エチニル基などが含まれる。これらの基は、置換されていてもよい。置換基は、特に限定されないが、好ましくは、アミノ基、水酸基、ＳＨ基、ハロゲン、カルボキシル基、ニトロ基等からなる群から選択される。 iii) substituted or unsubstituted alkyl group, alkenyl group or alkynyl group having 2 to 10 carbon atoms The alkyl group, alkenyl group or alkynyl group having 2 to 10 carbon atoms may be linear or branched, and in particular It is not limited. Preferably, a methyl group, an ethyl group, a propynyl group, an ethylene group, an ethynyl group and the like are included. These groups may be substituted. The substituent is not particularly limited, but is preferably selected from the group consisting of amino group, hydroxyl group, SH group, halogen, carboxyl group, nitro group and the like.
　ｉｖ）窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環
　Ｒ１及び／又はＲ２は、１又は複数のヘテロ環であってもよい。ヘテロ環は、チエニル基、チアゾリル基、イミダゾリル基、フラニル基、あるいはこれらの誘導体等から選択される、５員のヘテロ環である。好ましくは、２−チエニル基、２−チアゾリル基、２−イミダゾリル基、２，２’−ビチエン−５−イル基、２−（２−チアゾリル）チエン−５−イル基、５−（２−チエニル）チアゾール−２−イル基、及び２，２’，５’，２’’−ターチエン−５−イル基からなる群より選択される基である。 iv) one or more 5-membered heterocycles, 1 or more 6-membered heterocycles, 1 or more heterocycles, 1 or more aromatic rings R 1 and / or R containing nitrogen or sulfur atoms 2 may be one or more heterocycles. The heterocycle is a 5-membered heterocycle selected from a thienyl group, a thiazolyl group, an imidazolyl group, a furanyl group, or a derivative thereof. Preferably, 2-thienyl group, 2-thiazolyl group, 2-imidazolyl group, 2,2′-bithien-5-yl group, 2- (2-thiazolyl) thien-5-yl group, 5- (2-thienyl) ) A group selected from the group consisting of a thiazol-2-yl group and a 2,2 ′, 5 ′, 2 ″ -tert-en-5-yl group.
　６員ヘテロ環の例としては、ピラニル基、ピリジル基、ピリミジル基、等が挙げられる。複素環ヘテロ環の例としては、プリン、１−デアザプリン、キノリン等が挙げられる。
　芳香族環の例としては、フェニル基、ナフチル基等が挙げられる。 Examples of 6-membered heterocycles include pyranyl group, pyridyl group, pyrimidyl group, and the like. Examples of the heterocyclic ring include purine, 1-deazapurine, quinoline and the like.
Examples of the aromatic ring include a phenyl group and a naphthyl group.
　ヘテロ環、複素環ヘテロ環や芳香族環の数は、特に限定されないが、好ましくは１ないし３である。より好ましくは１又は２である。
　ｖ）糖、糖鎖、アミノ酸、ペプチド
　糖は、特に限定されない。例えば、グルコース、アラビノース、フラノース等が含まれる。リボースまたはデオキシリボースも糖の一種である。 The number of heterocycles, heterocycles and aromatic rings is not particularly limited, but is preferably 1 to 3. More preferably, it is 1 or 2.
v) Sugar, sugar chain, amino acid, peptide Sugar is not particularly limited. For example, glucose, arabinose, furanose and the like are included. Ribose or deoxyribose is also a kind of sugar.
　糖鎖は、特に限定されない、例えば、スクロース、ラクトース等が含まれる。
　アミノ酸も、特に限定されない。例えば、グリシン、アラニン、フェニルアラニン等が含まれる。 The sugar chain is not particularly limited, and examples thereof include sucrose and lactose.
Amino acids are not particularly limited. For example, glycine, alanine, phenylalanine and the like are included.
　ペプチドの種類は、特に限定されない。好ましくは、ポリペプチドは、約２ないし１０個のアミノ酸残基からなる。好ましいペプチドは例えば、フェニルアラニル−グリシンなどである。また、ペプチド核酸等の非天然型ペプチドも含まれる。 The type of peptide is not particularly limited. Preferably, the polypeptide consists of about 2 to 10 amino acid residues. A preferred peptide is, for example, phenylalanyl-glycine. In addition, non-natural peptides such as peptide nucleic acids are also included.
　ｖｉ）リンカーを介して結合した蛍光性分子
　リンカーの種類は特に限定されず、当業者は適宜採用可能である。リンカーは限定されるわけではないが、好ましくは、下記の化学式ＶＩＩおよびＶＩＩＩ： vi) Fluorescent molecule bonded through a linker The type of linker is not particularly limited, and those skilled in the art can appropriately employ it. The linker is not limited, but preferably the following chemical formulas VII and VIII:
Figure JPOXMLDOC01-appb-C000042
Figure JPOXMLDOC01-appb-C000042
　［式ＶＩＩ中、ｎは１ないし１２の整数から選択される］
及び [In the formula VII, n is selected from an integer of 1 to 12]
as well as
Figure JPOXMLDOC01-appb-C000043
Figure JPOXMLDOC01-appb-C000043
　［式ＶＩＩＩ中、ｍ及びｌは、１ないし１２の整数から各々独立に選択される］
からなる群より選択される。
　式ＶＩＩ及びＶＩＩＩにおいて、ｎ、ｍ、ｌは各々好ましくは１−７、より好ましくは５である。 [In Formula VIII, m and l are each independently selected from an integer of 1 to 12]
Selected from the group consisting of
In formulas VII and VIII, n, m and l are each preferably 1-7, more preferably 5.
　蛍光性分子の種類は特に限定されない。好ましくは、インドカルボシアニン（Ｃｙ３）、インドジカルボシアニン（Ｃｙ５）、５−カルボキシフルオレセイン（５−ＦＡＭ）、６−カルボキシフルオレセイン（６−ＦＡＭ）、５−カルボキシテトラメチルローダミン（５−ＴＡＭＲＡ）、６−カルボキシテトラメチルローダミン（６−ＴＡＭＲＡ）、５−ジメチルアミノナフタレン−１−スルホン酸（ＤＡＮＳＹＬ）、５−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（５−ＨＥＸ）、６−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（６−ＨＥＸ）、５−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（５−ＴＥＴ）、６−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（６−ＴＥＴ）、５−カルボキシ−Ｘ−ローダミン（５−ＲＯＸ）、及び６−カルボキシ−Ｘ−ローダミン（６−ＲＯＸ）からなる群から選択される。より好ましくは、インドカルボシアニン（Ｃｙ３）である。 The type of fluorescent molecule is not particularly limited. Preferably, indocarbocyanine (Cy3), indodicarbocyanine (Cy5), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA), 6-carboxytetramethylrhodamine (6-TAMRA), 5-dimethylaminonaphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (5 -HEX), 6-carboxy-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein (6-HEX), 5-carboxy-2', 4,7,7'-tetrachlorofluorescein ( 5-TET), 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein (6- ET), is selected from the group consisting of 5-carboxy -X- rhodamine (5-ROX), and 6-carboxy -X- rhodamine (6-ROX). More preferred is indocarbocyanine (Cy3).
　２．消光される蛍光を有する物質
　本発明の式Ｉで示す２−ニトロピロール構造を有する消光剤の消光作用によって消光される蛍光を有する物質の種類は特に限定されない。 2. Substance having fluorescence to be quenched The type of substance having fluorescence that is quenched by the quenching action of the quencher having a 2-nitropyrrole structure represented by Formula I of the present invention is not particularly limited.
　蛍光を有する物質は、例えば、蛍光性人工塩基、蛍光色素などの蛍光性分子等任意の物質が含まれる。
　式Ｉで示す２−ニトロピロール構造は、好ましくは以下の塩基と対合を形成する（特願２００９−２３２８５１）。 Examples of the substance having fluorescence include arbitrary substances such as fluorescent molecules such as fluorescent artificial bases and fluorescent dyes.
The 2-nitropyrrole structure represented by the formula I preferably forms a pair with the following base (Japanese Patent Application No. 2009-232851).
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓ）；
　７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）；
　７−（２，２’，５’，２’’−ターチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓｓ）；
　２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）；
　２−アミノ−６−（２，２’−ビチエン−５−イル）プリン−９−イル基（ｓｓ）；
　２−アミノ−６−（２，２’，５’，２’’−ターチエン−５−イル）プリン−９−イル基（ｓｓｓ）；
　４−（２−チエニル）−ピロロ［２，３−ｂ］ピリジン−１−イル基（ｄＤｓａ）；
　４−（２，２’−ビチエン−５−イル）−ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｓ）；
　４−［２−（２−チアゾリル）チエン−５−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｖ）；
　４−（２−チアゾリル）−ピロロ［２，３−ｂ］ピリジン−１−イル基（ｄＤｖａ）；
　４−［５−（２−チエニル）チアゾール−２−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｖａｓ）；及び
　４−（２−イミダゾリル）−ピロロ［２，３−ｂ］ピリジン−１−イル基（ｄＤｉａ）。 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds);
7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss);
7- (2,2 ′, 5 ′, 2 ″ -tertien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dsss);
2-Amino-6- (2-thienyl) purin-9-yl group (s);
2-amino-6- (2,2′-bithien-5-yl) purin-9-yl group (ss);
2-amino-6- (2,2 ′, 5 ′, 2 ″ -tert-en-5-yl) purin-9-yl group (sss);
4- (2-thienyl) -pyrrolo [2,3-b] pyridin-1-yl group (dDsa);
4- (2,2′-bithien-5-yl) -pyrrolo [2,3-b] pyridin-1-yl group (Dsas);
4- [2- (2-thiazolyl) thien-5-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dsav);
4- (2-thiazolyl) -pyrrolo [2,3-b] pyridin-1-yl group (dDva);
4- [5- (2-thienyl) thiazol-2-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dvas); and 4- (2-imidazolyl) -pyrrolo [2,3-b ] Pyridin-1-yl group (dDia).
　上記のうち、Ｄｓｓ、Ｄｓｓｓ、ｓｓ、ｓｓｓ、Ｄｓａｓ、Ｄｓａｖ及びＤｖａｓは蛍光性塩基である。これらは本発明の式Ｉの消光剤と塩基対を形成すると、蛍光強度が低下するか、あるいは消光する。 Among the above, Dss, Dsss, ss, sss, Dsas, Dsav and Dvas are fluorescent bases. When these form a base pair with the quencher of formula I of the present invention, the fluorescence intensity decreases or is quenched.
　式Ｉの消光剤と直接塩基対を形成しない蛍光を有する物質であっても、式Ｉの消光剤と塩基対を形成する上記人工塩基の近くに存在する場合には、本願発明の消光剤の影響を受けうる。例えば、蛍光を有する人工塩基が上記人工塩基（例えば、ｓ）と同一の一本鎖あるいは二本鎖、あるいは三本鎖核酸上で近接して存在する（例えばとなり同士で隣接する）場合、あるいは、上記人工塩基に結合している蛍光性分子の場合などは、式Ｉの消光剤と塩基対を形成する上記人工塩基の塩基対の形成により、蛍光を有する物質が本発明の消光剤の近傍に存在することとなり、消光の影響を受けうる。 Even if the substance having fluorescence that does not directly form a base pair with the quencher of formula I is present near the artificial base that forms a base pair with the quencher of formula I, Can be affected. For example, when an artificial base having fluorescence is present on the same single-stranded or double-stranded or triple-stranded nucleic acid as the above-mentioned artificial base (for example, s) (for example, adjacent to each other), or In the case of a fluorescent molecule bound to the artificial base, a substance having fluorescence is formed in the vicinity of the quencher of the present invention by forming a base pair of the artificial base that forms a base pair with the quencher of formula I. Can be affected by quenching.
　上述した式Ｉと塩基対を形成する人工塩基以外にも、蛍光性の核酸塩基類自体として、２−アミノプリン、エテノアデノシンなどが知られている。
ＩＩ．人工塩基対の形成を検出する方法
　本発明はまた、人工塩基対を検出する方法を提供する。本発明の方法は、
１．　　　　　　　　式ＩＩ In addition to the artificial base that forms a base pair with Formula I described above, 2-aminopurine, ethenoadenosine, and the like are known as fluorescent nucleobases themselves.
II. Method for Detecting Artificial Base Pair Formation The present invention also provides a method for detecting an artificial base pair. The method of the present invention comprises:
1. Formula II
Figure JPOXMLDOC01-appb-C000044
Figure JPOXMLDOC01-appb-C000044
　［式ＩＩにおいて、Ｒ２は、
　水素、水酸基、ＳＨ基、ハロゲン、
　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
　糖、糖鎖、アミノ酸、ペプチド、
　リンカーを介して結合した蛍光性分子、
からなる群から選択される基である］
で表される消光性人工塩基を有するヌクレオシドまたはヌクレオチド、あるいは、
２）蛍光共鳴エネルギー転移（ＦＲＥＴ）や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するヌクレオシドまたはヌクレオチド
のいずれか、あるいは双方を用いることを特徴とする。 [In Formula II, R 2 is
Hydrogen, hydroxyl group, SH group, halogen,
A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
Sugar, sugar chain, amino acid, peptide,
A fluorescent molecule bound via a linker,
A group selected from the group consisting of]
A nucleoside or nucleotide having a quenching artificial base represented by:
2) Either a natural base modified with self-quenching, an artificial base, or a nucleoside or nucleotide having a base analog that can be a donor such as fluorescence resonance energy transfer (FRET) or static quenching, Alternatively, both are used.
　式ＩＩの人工塩基の利用
　本発明の式ＩＩの消光性人工塩基のピロール環の窒素原子は、リボースまたはデオキシリボースに結合して、ヌクレオシドまたはヌクレオチドを構成する。本発明の式ＩＩの人工塩基は、Ｄｓ、Ｄｓｓ、Ｄｓｓｓ、ｓ、ｓｓ、ｓｓｓ、ｄＤｓａ、Ｄｓａｓ、Ｄｓａｖ、ｄＤｖａ、Ｄｖａｓ、及び、ｄＤｉａ等の人工塩基と人工塩基対を形成する（特願２００９−２３２８５１）。式ＩＩの消光性人工塩基とこれらの人工塩基との塩基対の形成により、塩基対を形成した蛍光性人工塩基、あるいは、付近に存在する蛍光性塩基若しくは蛍光性分子の蛍光強度が変化、あるいは消光する。本発明の方法はその変化を利用して人工塩基対の形成を検出する。 Use of Artificial Base of Formula II The nitrogen atom of the pyrrole ring of the quenching artificial base of formula II of the present invention forms a nucleoside or nucleotide by binding to ribose or deoxyribose. The artificial base of the formula II of the present invention forms an artificial base pair with artificial bases such as Ds, Dss, Dsss, s, ss, sss, dDsa, Dsas, Dsav, dDva, Dvas, and dDia (Japanese Patent Application 2009). -232851). Due to the formation of a base pair between the quenching artificial base of formula II and these artificial bases, the fluorescence intensity of the fluorescent artificial base forming the base pair, or the fluorescent base or fluorescent molecule present in the vicinity changes, or Extinguish. The method of the present invention uses this change to detect the formation of artificial base pairs.
　特に、上記のうち、Ｄｓｓ、Ｄｓｓｓ、ｓｓ、ｓｓｓ、Ｄｓａｓ、Ｄｓａｖ及びＤｖａｓは蛍光性塩基であり、式ＩＩの化合物と塩基対を形成することにより、人工塩基の蛍光が低下する、あるいは消光する。 In particular, among the above, Dss, Dsss, ss, sss, Dsas, Dsav and Dvas are fluorescent bases, and by forming a base pair with the compound of formula II, the fluorescence of the artificial base is reduced or quenched. .
　あるいは、本発明の消光性人工塩基に蛍光性分子を結合すると、本発明の消光性人工塩基の消光性により蛍光性分子の蛍光強度が低下する。これは蛍光性分子が消光性人工塩基と溶液中でスタッキングすることにより効率よく消光されるためと考えられる。そして、この蛍光性分子を結合した消光性人工塩基が、上記人工塩基との間で人工塩基対が形成し、核酸中に取り込まれると、蛍光性分子と消光性人工塩基との間のスタッキングが解消されるので、蛍光色素の蛍光強度が増す。この性質を利用して、人工塩基対の形成を検出することが可能である。 Alternatively, when a fluorescent molecule is bound to the quenching artificial base of the present invention, the fluorescence intensity of the fluorescent molecule is lowered due to the quenching property of the quenching artificial base of the present invention. This is considered to be because the fluorescent molecule is efficiently quenched by stacking with a quenching artificial base in a solution. Then, when the quenching artificial base to which the fluorescent molecule is bound forms an artificial base pair with the artificial base and is incorporated into the nucleic acid, stacking between the fluorescent molecule and the quenching artificial base occurs. Since it is eliminated, the fluorescence intensity of the fluorescent dye increases. Using this property, it is possible to detect the formation of artificial base pairs.
　蛍光共鳴エネルギー転移（ＦＲＥＴ）や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの利用
　本発明は、蛍光共鳴エネルギー転移（ＦＲＥＴ）や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するヌクレオシドまたはヌクレオチドを利用して、人工塩基対を検出する方法も含む。 Use of Fluorescence Resonance Energy Transfer (FRET), Static Quenching, etc. The present invention has a self-quenching property that can be a donor such as fluorescence resonance energy transfer (FRET) or static quenching. It also includes a method for detecting an artificial base pair using a nucleoside or nucleotide having a natural base modification, an artificial base, or a base analog.
　「蛍光共鳴エネルギー転移（ＦＲＥＴ）」は、ある蛍光分子から他の分子へ、共鳴により励起エネルギーが移動する現象を意味する。エネルギーを与える分子はドナー（供与体）、受け取る分子はアクセプター（受容体）と呼ばれる。ＦＲＥＴが生じると、エネルギーを失ったドナーは基底状態に戻り、同時にエネルギーを受け取ったアクセプターは励起状態となる。従って、ドナーの蛍光は弱まり、アクセプターが蛍光分子であればその蛍光が観察される。アクセプターが消光分子であれば、ドナーが単独の場合には観察されていた蛍光がＦＲＥＴにより観察されなくなる。ＦＲＥＴによるタンパク質の検出、核酸の検出の一般的な方法は公知である。 “Fluorescence resonance energy transfer (FRET)” means a phenomenon in which excitation energy is transferred from one fluorescent molecule to another by resonance. A molecule that gives energy is called a donor, and a molecule that receives energy is called an acceptor. When FRET occurs, the donor that has lost energy returns to the ground state, and at the same time, the acceptor that has received energy is in an excited state. Therefore, the fluorescence of the donor is weakened, and if the acceptor is a fluorescent molecule, the fluorescence is observed. If the acceptor is a quenching molecule, the fluorescence observed when the donor is used alone is not observed by FRET. General methods for protein detection and nucleic acid detection by FRET are known.
　ＦＲＥＴが生じるためには、以下の３条件を満たす必要がある。ｉ）ドナーの蛍光スペクトルとアクセプターの吸収スペクトルに重なりがあること。スペクトルの重なり範囲は大きい方が望ましいが、必ずしも完全に重なっている必要はない。ｉｉ）ドナーとアクセプターが物理的に近距離に存在すること。ＦＲＥＴが５０％の確率で生じる距離は３ｎｍないし６ｎｍと考えられており、ＦＲＥＴの効率はこの距離の変化に対して敏感に変化する。ｉｉｉ）ドナーとアクセプターの相対的な向きが適切であること。 In order for FRET to occur, the following three conditions must be satisfied. i) There is an overlap between the fluorescence spectrum of the donor and the absorption spectrum of the acceptor. Although it is desirable that the spectrum overlap range is large, it is not always necessary to completely overlap. ii) The donor and acceptor are physically close to each other. The distance at which FRET occurs with a probability of 50% is considered to be 3 nm to 6 nm, and the efficiency of FRET changes sensitively to changes in this distance. iii) The relative orientation of the donor and acceptor is appropriate.
　本発明の方法は、蛍光共鳴エネルギー転移（ＦＲＥＴ）の供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を利用するものである。また、消光の過程には、ＦＲＥＴ以外にエキシマーなどの励起二量体の形成による静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）も含まれる。人工塩基対の形成により、これらの自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体がアクセプターの近傍に存在した場合、これらの供与体に特有の波長のエネルギーを与えて励起することにより、これらの供与体からアクセプターにエネルギーが供与され、アクセプターが本来は蛍光を発しないはずの波長のエネルギーで蛍光を発する。 The method of the present invention utilizes a self-quenching natural base modification, artificial base, or base analog that can be a donor of fluorescence resonance energy transfer (FRET). In addition, the quenching process includes static quenching due to the formation of excimers such as excimers in addition to FRET. When these natural base modifiers, artificial bases, or base analogs having self-quenching properties are present in the vicinity of the acceptor due to the formation of an artificial base pair, these donors are excited by giving energy of a specific wavelength. As a result, energy is donated from these donors to the acceptor, and the acceptor emits fluorescence with energy of a wavelength that should not emit fluorescence.
　限定するわけではないが、「自己消光性を有する人工塩基」としては、１以上の隣接するｓ、例えば、同一核酸上で隣接する２以上のｓ、ｓｓ、Ｄｓｓ、Ｄｓｓｓなどが含まれる。 Although not necessarily limited, “artificial base having self-quenching” includes one or more adjacent s, for example, two or more adjacent s, ss, Dss, and Dsss on the same nucleic acid.
　好ましくは、同一核酸上で隣接する２以上のｓである。「自己消光性を有する天然塩基修飾体」とは、非限定的に、上記自己消光性を有する人工塩基（例えばｓ）を１以上結合した天然塩基（例えば、ｓを結合したウラシル、ｓを２個結合したシトシン、Ｄｓｓを結合したウラシル）、などが含まれる。「自己消光性を有する塩基類似体」とは、例えば、サイズエキスパンデッド塩基類似体（ｓｉｚｅ−ｅｘｐａｎｄｅｄ　ｂａｓｅ　ａｎａｌｏｇｓ）二量体、２−アミノプリン二量体などが含まれる。 Preferably, it is 2 or more adjacent s on the same nucleic acid. “Natural base modified product having self-quenching property” means, but is not limited to, a natural base (for example, uracil to which s is bound, s to 2) to which at least one artificial base having self-quenching properties (for example, s) is bound. Single-linked cytosine, uracil bonded with Dss), and the like. Examples of the “base analog having self-quenching property” include a size-expanded base analog dimer, a 2-aminopurine dimer, and the like.
　ここで検出される人工塩基対は、好ましくは上記式ＩＩで表される消光性人工塩基とその相補的な人工塩基との間の塩基対である。しかしながら必ずしもそれに限定されない。蛍光共鳴エネルギー転移（ＦＲＥＴ）や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）の供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するヌクレオシドまたはヌクレオチドを利用する方法であれば、その他の公知の人工塩基対であっても本発明の範囲に含まれる。例えば、ｓ−ｙ塩基対（ｓ：２−アミノ−６−チエニルプリン、ｙ：ピリジン−２−オン）、ｖ−ｙ塩基対（ｖ：２−アミノ−６−チアゾリルプリン）、ｓ−Ｐａ塩基対（Ｐａ：ピロール−２−カルバルデヒド）、Ｄｓ−Ｐａ塩基対（Ｄｓ：７−（２−チエニル）−イミダゾ［４，５−ｂ］ピリジン）、Ｐａ−Ｑ塩基対（Ｑ：９−メチルイミダゾ［（４，５）−ｂ］ピリジン）、ｉｓｏＧ−ｉｓｏＣ、５ＳＩＣＳ−ＭＭＯ２、５ＮａＭなどの人工塩基対も、本発明のＦＲＥＴを利用した方法により検出することが可能である。 The artificial base pair detected here is preferably a base pair between the quenching artificial base represented by the above formula II and its complementary artificial base. However, it is not necessarily limited thereto. Any method that uses a natural base modifier, an artificial base, or a nucleoside or nucleotide having a base analog that has a self-quenching property that can serve as a donor for fluorescence resonance energy transfer (FRET) or static quenching (static quenching) Other known artificial base pairs are also included in the scope of the present invention. For example, sy base pair (s: 2-amino-6-thienylpurine, y: pyridin-2-one), vy base pair (v: 2-amino-6-thiazolyl purine), s-Pa base pair (Pa: pyrrole-2-carbaldehyde), Ds-Pa base pair (Ds: 7- (2-thienyl) -imidazo [4,5-b] pyridine), Pa-Q base pair (Q: 9-methylimidazo) Artificial base pairs such as [(4,5) -b] pyridine), isoG-isoC, 5SICS-MMO2, and 5NaM can also be detected by the method using FRET of the present invention.
［図１］図１は、消光性塩基（Ｐｎ、Ｐｘ）とその相補性塩基（ＤｓやＤｓｓ）との人工塩基対の例を示す。例は、Ｐｎと蛍光性人工塩基（Ｄｓｓ）、ならびにＰｘとＤｓからなる人工塩基対である。
［図２］図２は、本発明の実施例に使用した、消光性人工塩基Ｐｎとその４’誘導体の構造を示す。
［図３］図３は、本発明の実施例に使用した、消光性人工塩基Ｐｘとその誘導体の構造を示す。
［図４］図４は、本発明の方法に使用する、Ｐｎ又はＰｘと相補性を有する人工塩基の例として、Ｄｓ、並びに、蛍光性人工塩基Ｄｓｓ、Ｄｓｓｓ、Ｄｓａｖの構造を示す。
［図５］図５は、ＮＨ２−ｈｘ−ｄＰｘＴＰからＣｙ３−ｈｘ−ｄＰｘＴＰの合成を示す。反応条件は、以下の通りである。１００ｍＭ　ＮａＨＣＯ３中、ＮＨ２−ｈｘ−ｄＰｘＴＰ（８．４μｍｏｌ）、Ｎａ２ＣＯ３緩衝液（ｐＨ８．５）（５００μｌ）、ＤＭＦ（３００μｌ）中、Ｃｙ３（７．６３μｍｏｌ）　Ｎ−ヒドロキシスクシニミジルエステル、室温１２時間
［図６］図６は、蛍光性人工塩基Ｄｓｓまたはｓを結合した天然型塩基のアミダイト試薬の構造を示す。
　図６中の化合物は、左からＤｓｓ−ｈｘ−ｄＵアミダイト、ｓ−ｈｘ−ｄＵアミダイト、そして、ｓ２−ｈｘ−ｄＣアミダイトである。
［図７］図７は、人工塩基Ｐｎによる相補鎖中の蛍光性人工塩基Ｄｓｓの消光を示す。１０ｍＭ　リン酸ナトリウム（ｐＨ７．０）、１００ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＡＴ中の各ＤＮＡ溶液（５μＭ）を３６５ｎｍの照射により撮影した。蛍光性人工塩基（Ｄｓｓ）を含む一本鎖のオリゴヌクレオチド（１２−ｍｅｒ）を消光性人工塩基（Ｐｎ）を含む相補鎖のオリゴヌクレオチド（１２−ｍｅｒ）と二本鎖を形成させると、Ｄｓｓの蛍光がＰｎにより消光される（図７、左から２番目）。天然型塩基（Ｔなど）や人工塩基（ＤｓｓやＤｓ）と塩基対を形成してもＤｓｓの蛍光は消光されない（図７、左から３番目−５番目）。
［図８］図８は、図７の実験の各ＤＮＡ断片の蛍光スペクトルを示す。Ｄｓｓを含む一本鎖ＤＮＡ（５’−ＧＧＴＡＡＣＤｓｓＡＴＧＣＧ−３’）、Ｄｓｓ−Ｐｎ，Ｄｓｓ−Ｄｓｓ，Ｄｓｓ−Ｄｓ，Ｄｓｓ−Ｔ塩基対のそれぞれを含む二本鎖ＤＮＡ（５’−ＧＧＴＡＡＣＮＡＴＧＣＧ−３’（Ｎ＝Ｄｓｓ）及び５’−ＣＧＣＡＴＮ’ＧＴＴＡＣＣ−３’（Ｎ’＝Ｄｓｓ，Ｄｓ，Ｐｎ又はＴ）の５μＭ　ＤＮＡ溶液の蛍光スペクトル（励起波長：３８５ｎｍ、２５℃）の結果である。図８に示されるようにＤｓｓの蛍光はＰｎにより５分の１程度に消光された。
［図９］図９は、Ｐｎの消光作用を調べた結果を示す。具体的には、蛍光性人工塩基Ｄｓｓの２’−デオキシリボヌクレオシド　５’‐三リン酸誘導体（ｄＤｓｓＴＰ）の水溶液での消光をｄＰｎＴＰの濃度依存性として調べた。Ａ：　Ｐｎのデオキシリボヌクレオシド三リン酸（ｄＰｎＴＰ）の濃度依存による蛍光性人工塩基Ｄｓｓのデオキシリボヌクレオシド三リン酸（ｄＤｓｓＴＰ，５μＭ）の蛍光強度の変化。Ｂ：　ｄＰｎＴＰと天然型塩基の三リン酸とのＤｓｓに対する消光性を比較した結果を示す。Ｐｎならびに天然型塩基のデオキシリボヌクレオシド三リン酸による、蛍光性塩基ｄＤｓｓＴＰ（５μＭ）の消光のＳｔｅａｄｙ−ｓｔａｔｅ　Ｓｔｅｒｎ−Ｖｏｌｍｅｒ　ｐｌｏｔ。１００ｍＭ　ＮａＣｌ、１０ｍＭ　リン酸ナトリウム（ｐＨ７．０）、０．１ｍＭ　ＥＤＴＡの溶液中、２０℃で、３７０ｎｍの励起による蛍光を測定し、以下の式よりＳｔｅｒｎ−Ｖｏｌｍｅｒ定数（Ｋｓｖ）を算出した。
　Ｓｔｅｒｎ−Ｖｏｌｍｅｒ　ｅｑｕａｔｉｏｎ　：　Ｆ０／Ｆ１＝１＋ＫＳＶ［Ｑ］
　［Ｆ０及びＦ１、：　各々消光剤を伴う（Ｆ１）又は伴わない（Ｆ０）場合の蛍光強度　［Ｑ］：　消光剤の濃度］グアニン塩基は消光性を有することが知られているが、Ｐｎはさらに強い消光性を示す（図９Ｂ）。
［図１０］図１０は、Ｐｎとその各種誘導体（図２、図３）、ならびにＰｘによるＤｓｓの蛍光の消光特性を示す。具体的には、エタノール、２５℃で、３８５ｎｍの励起による蛍光を測定し、Ｐｎの各種誘導体とＰｘのデオキシリボヌクレオシド（Ａ：２．５ｍＭ、Ｂ：５ｍＭ）共存下での、ｄＤｓｓ（５μＭ）の蛍光強度の変化を調べた。いずれの誘導体もＰｎよりもさらに強い消光特性を示した。
［図１１］図１１は、大腸菌由来のＤＮＡポリメラーゼＩのクレノウ断片による、Ｐｎを含む鋳型ＤＮＡとｄＤｓｓＴＰを用いたプライマー伸長反応を調べた結果を示す。２００ｎＭの鋳型ＤＮＡ、１０μＭのｄＣＴＰとｄＴＴＰ、０．１Ｕ／μｌのクレノウ断片に、各濃度のｄＤｓｓＴＰを加えて、３７℃、３分反応を行い、変性ゲル電気泳動で解析した。ｄＡＴＰとｄＧＴＰを加えていないので、伸長反応が進んだ際の生成物は鋳型のＣの手前で止まった３３−ｍｅｒになる。図１１は、ｄＤｓｓＴＰが鋳型中のＰｎに相補して、相補鎖ＤＮＡに取り込まれることを示している。既にｄＤｓｓＴＰは鋳型中のＰａに対しても取り込まれることを明らかにしているが（Ｊ．Ａｍ．Ｃｈｅｍ．Ｓｏｃ．，１３２：４９８８−４９８９，２０１０）、Ｐｎの方がＤｓｓの取り込み効率が高くなることが分かった。また、ｄＤｓｓＴＰの濃度を高くするとＰｎやＰａに対してＤｓｓが取り込まれた後の伸長反応が阻害されるが、ｄＤｓｓＴＰの濃度を下げることにより、プライマー伸長反応が効率よく進行した。
［図１２］図１２は、Ｄｓｓ−Ｐｘ塩基対を用いたＤｓを含むＤＮＡのＰＣＲ増幅を調べた結果を示す。Ｄｓを含むＤＮＡ（５５−ｍｅｒ）をｄＤｓｓＴＰとＮＨ２−ｈｘ−ｄＰｘＴＰ、ならびに天然型塩基の基質を用いて２０サイクルのＰＣＲ増幅を行い、変性ゲル電気泳動後、ＳＹＢＲ　Ｇｒｅｅｎ　ＩＩで染色後に生成物を解析した。Ｄｓを含む鋳型ＤＮＡ（５５−ｍｅｒ、Ｓ２）を用いて、天然型塩基の基質（ｄＮＴＰｓ）にｄＤｓｓＴＰとＮＨ２−ｈｘ−ｄＰｘＴＰを加えて、ＰＣＲを行った結果である。天然型塩基のみの鋳型ＤＮＡと同様に人工塩基を含む鋳型ＤＮＡ−Ｓ２も増幅されることが分かった。Ｄｓｓ−ＰｎならびにＤｓｓ−Ｐｘ塩基対は、ＰＣＲにおいても効率よく機能する。
［図１３］図１３は、Ｄｓｓ−Ｐｘ塩基対を用いたＰＣＲ増幅後のＤＮＡの配列決定を行った結果を示す。Ｄｓを含むＤＮＡ（５５−ｍｅｒ）をｄＤｓｓＴＰとＮＨ２−ｈｘ−ｄＰｘＴＰ、ならびに天然型塩基の基質を用いて１５サイクルのＰＣＲ増幅を行い、従来法で増幅産物の配列決定を行った。増幅されたＤＮＡ中にはＤｓｓとＮＨ２−ｈｘ−Ｐｘが９９％以上保持されていることが分かった。本配列決定はこれまでに本願発明者らが開発した方法を用いている（Ａｎ　ｕｎｎａｔｕｒａｌ　ｈｙｄｒｏｐｈｏｂｉｃ　ｂａｓｅ　ｐａｉｒ　ｓｙｓｔｅｍ：ｓｉｔｅ−ｓｐｅｃｉｆｉｃ　ｉｎｃｏｒｐｏｒａｔｉｏｎ　ｏｆ　ｎｕｃｌｅｏｔｉｄｅ　ａｎａｌｏｇｓ　ｉｎｔｏ　ＤＮＡ　ａｎｄ　ＲＮＡ．Ｉ．Ｈｉｒａｏ，Ｍ．Ｋｉｍｏｔｏ，Ｔ．Ｍｉｔｓｕｉ，Ｔ．Ｆｕｊｉｗａｒａ，Ｒ．Ｋａｗａｉ，Ａ．Ｓａｔｏ，Ｙ．Ｈａｒａｄａ，Ｓ．Ｙｏｋｏｙａｍａ，Ｎａｔｕｒｅ　Ｍｅｔｈｏｄｓ，３，７２９−７３５（２００６）；Ａｎ　Ｕｎｎａｔｕｒａｌ　ｂａｓｅ　ｐａｉｒ　ｓｙｓｔｅｍ　ｆｏｒ　ｅｆｆｉｃｉｅｎｔ　ＰＣＲ　ａｍｐｌｉｆｉｃａｔｉｏｎ　ａｎｄ　ｆｕｎｃｔｉｏｎａｌｉｚａｔｉｏｎ　ｏｆ　ＤＮＡ　ｍｏｌｅｃｕｌｅｓ．Ｍ．Ｋｉｍｏｔｏ，Ｒ．Ｋａｗａｉ，Ｔ．Ｍｉｔｓｕｉ，Ｓ．Ｙｏｋｏｙａｍａ，ａｎｄ　Ｉ．Ｈｉｒａｏ，Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，３７，ｅ１４（２００９））
［図１４］図１４は、Ｄｓｓ−Ｐｘ塩基対を用いたリアルタイムＰＣＲの原理を示した模式図である。ＤｓｓをＰＣＲのプライマー中に導入し、ｄＰｎＴＰあるいはｄＰｘＴＰを用いてＰＣＲを行うと、Ｄｓｓに相補してＰｎやＰｘが相補鎖中に取り込まれ、Ｄｓｓの蛍光が消光される。これを検出することによりリアルタイムＰＣＲが可能になる。
［図１５］図１５は、Ｄｓｓ−Ｐｘ塩基対を用いたリアルタイムＰＣＲを行った結果を示す。図１４で示したＤｓｓを含むプライマーを用いると、その相補鎖にｄＰｘＴＰが取り込まれ、Ｄｓｓの蛍光が消光される。これはＰＣＲの際にＤｓｓ−Ｐｘ塩基対が形成されて、Ｄｓｓの蛍光が消光され、リアルタイムＰＣＲへの応用が可能になる。
　反応混合液（２５μＬスケール）
　１×Ｔｉｔａｎｉｕｍ　Ｔａｑ　ＰＣＲ　ｂｕｆｆｅｒ
　１μＭ　０８０７３１−５’プライマー３　（配列番号１５）
　１μＭ　０９０９１４ａ−Ｐｌｅｘｏｒ−Ｄｓｓ１　（配列番号１６）
　２μＭ　ｄＰｘＴＰ
　２ｍＭ　ｄＮＴＰｓ
　１×Ｔｉｔａｎｉｕｍ　ＴａｑＤＮＡ　ｐｏｌｙｍｅｒａｓｅ
　２ａＭ（３コピー）−２ｆＭ（３００００コピー）　鋳型ＤＮＡ適用
　滅菌水で全量２５μＬとした。
　ＰＣＲ条件
　９４℃、２分→［９４℃、５秒−６８℃、４０秒］×５５サイクル
［図１６］図１６は、Ｄｓｓ−Ｐｎ塩基対を含むＤＮＡヘアピンの蛍光特性を調べた結果を示す。Ｄｓｓ−Ｐｎ塩基対を含むＤＮＡヘアピン（３４−ｍｅｒ）とＤｓｓを含む一本鎖ＤＮＡ（１２−ｍｅｒ）のそれぞれのＤｓｓの蛍光強度の温度依存性を測定した。それぞれのＤＮＡを１μＭ、２ｍＭ塩化マグネシウムを含む緩衝溶液中で測定した。ＤｓｓとＰｎを塩基対としてヘアピン核酸のステム領域に導入することにより、ヘアピン構造を形成しているとＤｓｓの蛍光がＰｎにより消光され、このヘアピンＤＮＡを熱変性させると、その過程でＤｓｓの蛍光強度が増加した。このＤｓｓ−Ｐｎ（あるいはＤｓｓ−Ｐｘ）塩基対の性質は、モレキュラービーコンに応用することが出来る。
［図１７］図１７は、Ｄｓｓ−Ｐｎ塩基対を含むモレキュラービーコンの可視化が可能になったことを示す。Ｄｓｓ−Ｐｎ塩基対を含むヘアピン型ビーコン（２６−ｍｅｒ）の蛍光をターゲットの一本鎖ＤＮＡ（７１−ｍｅｒ）の存在下、非存在下で観測した。それぞれのＤＮＡを１μＭ、１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０）・１００ｍＭ　ＮａＣｌ　・０．１ｍＭ　ＥＤＴＡ中で測定した。その結果、Ｄｓｓ−Ｐｎ塩基対を含むモレキュラービーコンのループ部分に相補するＤＮＡを認識して二本鎖を形成するとＰｎによるＤｓｓの蛍光消光が解消され、紫外線を照射することにより、Ｄｓｓの蛍光を裸眼で確認することが出来た。
［図１８］図１８は、Ｄｓｓ−Ｐｎ塩基対を含むモレキュラービーコンによる一塩基変異を検出した結果を示す。Ｄｓｓ−Ｐｎ塩基対を含む２種類のヘアピン型ビーコン（２６−ｍｅｒ）のそれぞれの蛍光を１塩基変異の２種類の標的配列のそれぞれの一本鎖ＤＮＡ（７１−ｍｅｒ）に加えて観測した。それぞれのＤＮＡを１μＭ、１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０）・１００ｍＭ　ＮａＣｌ・０．１ｍＭ　ＥＤＴＡ中で測定した。２種類の標的配列について各標的ＤＮＡに相補する配列をループ部分に有するモレキュラービーコンを作成することにより、１塩基の違いを、ハイブリダイゼーションによりＤｓｓの発光強度の違いとして、識別することが出来た。
［図１９］図１９は、蛍光色素Ｃｙ３をＰｘに結合させた基質として、Ｃｙ３−Ｐｘ／Ｄｓｓ塩基対を用いた可視化ＰＣＲの原理を示す。
［図２０］図２０は、図１９に記載の原理に基づき、Ｃｙ３−Ｐｘ／Ｄｓｓ塩基対を用いた可視化リアルタイムＰＣＲを行った例を示す。
　反応混合液（２５μＬスケール）
　１×Ｔｉｔａｎｉｕｍ　Ｔａｑ　ＰＣＲ　ｂｕｆｆｅｒ
　１μＭ　０８０７３１−５’プライマー３　（配列番号１５）
　１μＭ　０９０９１４ａ−Ｐｌｅｘｏｒ−Ｄｓｓ１　（配列番号１６）
　２μＭ　Ｃｙ３−ｈｘ−ｄＰｘＴＰ
　２ｍＭ　ｄＮＴＰｓ
　１×Ｔｉｔａｎｉｕｍ　ＴａｑＤＮＡ　ｐｏｌｙｍｅｒａｓｅ
　２ａＭ（３コピー）−２００ｆＭ（３００００００コピー）　鋳型ＤＮＡ適用
　滅菌水で全量２５μＬとした。
　ＰＣＲ条件
　９４℃、２分→［９４℃、５秒−６８℃、４０秒］×５５サイクル
　Ｃｙ３は３５０ｎｍ近辺の励起波長では発光しないので、Ｃｙ３を結合したＰｘの基質（Ｃｙ３−ｈｘ−ｄＰｘＴＰ）は３５０ｎｍのＵＶ照射では発光しない。Ｄｓｓ−Ｐｘの塩基対形成によりＣｙ３−ｈｘ−ｄＰｘＴＰがＤｓｓの相補鎖に取り込まれると、３５０−３９０ｎｍのＵＶ照射によりＤｓｓからＣｙ３への蛍光共鳴エネルギー転移（ＦＲＥＴ）が生じ発光する。これにより、Ｃｙ３の発光をオレンジフィルターを通して観察することにより、ＰＣＲ増幅されたＤＮＡを裸眼で検出することが出来る。
［図２１］図２１は、蛍光性分子（Ｃｙ３）を結合した消光性Ｐｘ塩基によるリアルタイムＰＣＲ法の原理を示した模式図である。蛍光性分子（例えばＣｙ３など）を消光性のＰｘ塩基に結合させると、蛍光性分子の蛍光が３０％程度消光する。これを基質（Ｃｙ３−ｈｘ−ｄＰｘＴＰ）にして、Ｄｓ塩基を導入したプライマーを用いてＰＣＲを行うとＣｙ３−ｈｘ−ｄＰｘがＤＮＡ中に取り込まれることで、Ｃｙ３の蛍光強度が増大する。この方法はリアルタイムＰＣＲに用いることができる（図２２）。
［図２２］図２２は、蛍光性分子（Ｃｙ３）を結合した消光性のＰｘ塩基を用いてリアルタイムＰＣＲを行った結果を示す。リアルタイムＰＣＲ装置（ストラタジーン、Ｍｘ３００５Ｐ）を用いて、Ｃｙ３−ｈｘ−ｄＰｘＴＰを基質として用いたリアルタイムＰＣＲ検出を行った。ＰＣＲ反応は、プライマー：各１μＭ、天然型塩基基質ｄＮＴＰ：各０．２ｍＭ、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ：２μＭで実施し、励起波長５４５ｎｍで５６８ｎｍの蛍光変化を検出した。５４５ｎｍの照射で直接、Ｃｙ３を励起するため図１９と図２０の応用例と異なり、この場合には遊離の基質のＣｙ３−ｈｘ−ｄＰｘＴＰも発光する。よってこの方法の場合は、図２０のように裸眼では識別できない。そこで、ＤＮＡ中にＣｙ３−ｈｘ−ｄＰｘＴＰが取り込まれた際の蛍光強度の増加の測定に、リアルタイムＰＣＲ装置を用いた。
［図２３］図２３は、蛍光性分子（Ｃｙ３）を結合した消光性のＰｘ塩基によるリアルタイムＰＣＲ産物をゲル電気泳動により検出した結果を示す。図２２で示したＰＣＲ産物は、Ｃｙ３が取り込まれているので、この産物をアガロースゲルで電気泳動すると、従来のＥｔＢｒやＳＹＢＲ　ＧｒｅｅｎなどのＤＮＡ染色用の色素を用いることなく、ゲル上でＰＣＲ産物をＣｙ３の蛍光で検出することができる。
　検出条件
　ＦＬＡ７０００バイオイメージングアナライザー
　（Ｃｙ３　ｍｏｄｅ）
　５３２ｎｍ　レーザー／Ｏ５８０　蛍光フィルター
　ＰＭＴ：　５００Ｖ
［図２４］図２４は、蛍光性分子（Ｃｙ３）ならびに蛍光性人工塩基ｓを含むＤＮＡの蛍光特性を調べた結果である。測定条件：１００ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＴＡを含む１０ｍＭ　リン酸ナトリウム（ｐＨ７．０）に、各種ＤＮＡ断片が５μＭとなるように調製し、２５４−３６５ｎｍの照射で発光を観察した。　レーン２は蛍光性人工塩基ｓを１つ含むＤＮＡ断の場合であり、ｓのみをＤＮＡ中に導入すると２５４‐３６５ｎｍの照射でｓの発光が起こる。しかし、ｓを２つ隣接してＤＮＡ中に導入するとその蛍光が消光する（レーン３）。天然型塩基だけからなるＤＮＡ断片にＣｙ３を結合させた場合、３６５ｎｍの照射では発光しない（レーン４）。しかしながら、ＤＮＡ中のＣｙ３近傍にｓを１つあるいは２つを導入すると、ｓとＣｙ３間でＦＲＥＴが起こり、Ｃｙ３の蛍光が観測されるようになる（レーン５−７）。具体的には、３６５ｎｍでｓを励起するとＣｙ３のオレンジ色の発光が観測される（レーン５，６）。さらに２つのｓを隣接してＤＮＡ断片中に導入すると、ｓ同士の消光により２５４‐３６５ｎｍで励起してもｓの発光はほとんど観測されないが（レーン３）、このＤＮＡ断片にＣｙ３を結合させるとＦＲＥＴが起こり、Ｃｙ３の発光が観測される（レーン７）。この現象を利用して、複製や転写によるＤＮＡの増幅の検出を裸眼で行うことができる。
［図２５］図２５は、蛍光性分子（Ｃｙ３）を結合した消光性のＰｘ塩基と蛍光性人工塩基ｓを組み合わせた可視化ＰＣＲ法の原理を示す。蛍光性人工塩基ｓを隣接して２つ導入するとｓの蛍光は完全に消光し、３５０ｎｍの照射で発光しない。それに近接するＤｓも３５０ｎｍで発光しない。２つの隣接するｓ及びその近傍にＤｓを含むプライマーとＣｙ３−ｈｘ−ｄＰｘＴＰを用いてＰＣＲを行うと、その相補鎖にＣｙ３−ｈｘ−Ｐｘが取り込まれる。２つのｓとＤｓが近い位置にあるため、ｓの励起波長である３６５ｎｍの照射でＦＲＥＴが起こり、近傍のＣｙ３などの蛍光色素が蛍光発光する（図２６）。これを用いてＰＣＲで増幅されたＤＮＡを裸眼で検出することができる（図２６、２７）。
［図２６］図２６は、蛍光性分子（Ｃｙ３）を結合した消光性のＰｘ塩基と蛍光性人工塩基ｓを組み合わせた可視化ＰＣＲ法の結果を示す。図２６にはＰＣＲを行い、ＰＣＲチューブを３５０ｎｍで照射し、裸眼であるいはオレンジ色のフィルターを通してＣｙ３の発光を調べた結果を示す。従来法では、ＰＣＲによるＤＮＡ増幅を裸眼で確認することは難しかった。即ち、従来の例えばＳＹＢＲ　Ｇｒｅｅｎを用いる方法は、リアルタイムＰＣＲで最も用いられる検出法であるが、図中右側のように裸眼での検出は難しい。これに対し、本発明の方法は、リアルタイムＰＣＲのみならず裸眼でもＰＣＲの検出が可能であった（図２６左側）。
［図２７ａ］図２７ａは、蛍光性分子（Ｃｙ３）を結合した消光性のＰｘ塩基と蛍光性人工塩基ｓを組み合わせた可視化ＰＣＲ法を行った結果を示す。標的ＤＮＡを３コピーから３００万コピー用いて、５５サイクルのＰＣＲを行い、ＰＣＲ産物を電気泳動により解析した結果は図２８に示している。本検出系では、電気泳動することなく、３６５ｎｍのＵＶ励起によるＣｙ３の蛍光の有無を反応チューブレベルで観察するだけで、３コピーのＤＮＡでも５５サイクルのＰＣＲ後、その増幅産物を裸眼で検出が可能であることを示している。
［図２７ｂ］図２７ｂは、蛍光性分子（Ｃｙ３）を結合した消光性Ｐｘ塩基と蛍光性人工塩基ｓを組み合わせた可視化ＰＣＲをリアルタイム定量ＰＣＲ装置により検出した結果を示す。
　反応混合物（２５μスケール）
　１μＭ　０８０７３１　５’ｐｒｉｍｅｒ　３
　１μＭ　Ｐｒｉｍｅｒ　２ｄ−Ｄｓ−ｓｓ３　３’ｐｒｉｍｅｒ
　２μＭ　Ｃｙ３−ｈｘ−ｄＰｘＴＰ
　２００μＭ　ｄＮＴＰｓ
　１×Ｔｉｔａｎｉｕｍ　Ｔａｑ　Ｂｕｆｆｅｒ
　１×Ｔｉｔａｎｉｕｍ　Ｔａｑ　ＤＮＡポリメラーゼ
　２ａＭ（３ｃｏｐｉｅｓ）~２００ｆＭ（３００００００ｃｏｐｉｅｓ）９８Ｇ　ｔｅｍｐｌａｔｅ
　ＰＣＲ条件：　９４℃−２分　→　［９４℃−５秒→６８℃−４０秒］×３０−５５サイクル
　本法はＤＮＡ中に取り込まれたＣｙ３‐ｈｘ−ＰｘのＣｙ３の蛍光強度の増加によりリアルタイムＰＣＲも可能である。
［図２７ｃ］図２７ｃは、図２７ｂの各ＰＣＲサイクルのＤＮＡ増幅産物を可視化した結果を示す。
［図２７ｄ］図２７ｄは、図２７ｃの各ＰＣＲチューブの蛍光強度を定量化した結果を示す。図２７ｄ１は、０、３−３００００コピーのそれぞれのＤＮＡをＰＣＲで増幅した時の各ＰＣＲサイクルにおける蛍光強度をプロットしたものである。図２７ｄ２は、３−３００００００コピーのそれぞれのＤＮＡを各ＰＣＲサイクルで増幅した時の蛍光強度をプロットした図である。
［図２８］図２８は、蛍光性分子（Ｃｙ３）を結合した消光性のＰｘ塩基と蛍光性人工塩基ｓを組み合わせたプライマーを用いたＰＣＲ（５５サイクル）による産物のゲル電気泳動上での検出を行った結果を示す。本発明の方法により図２７ａの可視化ＰＣＲ産物をアガロースゲル電気泳動することにより、３１２ｎｍあるいは５３２ｎｍの照射でＰＣＲ産物を検出することができた。３１２ｎｍの照射の場合にはｓからＣｙ３へのＦＲＥＴを検出しており、５３２ｎｍの照射の場合には、ＤＮＡ中に取り込まれたＣｙ３を直接励起した結果を示している。ＰＣＲ産物はＣｙ３で標識されているため、３１２ｎｍでｓを励起してＦＲＥＴにより、あるいは、５３２ｎｍで直接Ｃｙ３を励起することにより、ＰＣＲ産物をゲル上で観察することが出来た。
［図２９ａ］図２９ａは、蛍光性分子（ｓ塩基）を天然型塩基にリンカーを介して結合したヌクレオシド誘導体（図６、ｓ−ｈｘ−ｄＵ、（Ｕｓ））とＤｓ−Ｐｘ塩基対を用いたＰＣＲ産物の検出法を示した模式図である。図２５の可視化ＰＣＲ法では、隣接する２つの蛍光性人工塩基ｓを用いているが、代わりにこの蛍光性のｓを天然型塩基にリンカーを介して結合し、ＰＣＲ用のプライマー中にこの塩基を２つ隣接させたものが、図２９ａの態様である。
［図２９ｂ］図２９ｂは、蛍光性分子（ｓ塩基）を天然型塩基にリンカーを介して結合したヌクレオシド誘導体（図６、ｓ−ｈｘ−ｄＵ、（Ｕｓ））とＤｓ−Ｐｘ塩基対を用いたＰＣＲについて、使用する各プライマー、鋳型の配列、並びにＰＣＲの条件を示した図である。
［図２９ｃ］図２９ｃは、蛍光性分子（Ｃｙ３）を結合した消光性のＰｘ塩基と蛍光性人工塩基ｓ−ｈｘ−ｄＵを組み合わせた可視化ＰＣＲを行った結果を示す。ＤＮＡ中に取り込まれたＣｙ−ｈｘ−ＰｘのＣｙ３の蛍光強度の増加によりリアルタイムＰＣＲも可能となった。
［図２９ｄ］図２９ｄは、図２９ｃの各ＰＣＲサイクルのＤＮＡ増幅産物を可視化したものである。
［図３０］図３０は、ｓ−ｈｘ−ｄＵアミダイト試薬の化学合成である。
　条件：（ａ）ＣＢｒ４，ＰＰｈ３，ＣＨ２Ｃｌ２；
１．　　（ｂ）Ｋ２ＣＯ３，ＤＭＦ；
２．　　（ｃ）Ｐａｃ−Ｃｌ，ＨＯＢＴ，ピリジン，ＣＨ３ＣＮ；
３．　　（ｄ）ＤＭＴｒ−デオキシ−５−ヨードウリジン，Ｐｄ（ＰＰｈ３）４，ＣｕＩ，ＴＥＡ，ＤＭＦ；
４．　　（ｅ）ＮＣ（ＣＨ２）２Ｏ−Ｐ（Ｃｌ）Ｎ（ｉＰｒ）２，ＤＩＥＡ，ＴＨＦ
［図３１］図３１は、２分子の蛍光性塩基（ｓ）を天然型塩基にリンカーを介して結合したヌクレオシド誘導体（図６、ｓ２−ｈｘ−ｄＣ、（Ｃｓｓ））とＤｓ−Ｐｘ塩基対を用いたＰＣＲ産物の検出方法を示した模式図である。
［図３２］図３２は、蛍光性分子（Ｄｓｓ塩基）を天然型塩基にリンカーを介して結合したヌクレオチド誘導体（図６、Ｄｓｓ−ｈｘ−ｄＵ（ＵＤｓｓ））とＤｓ−Ｐｘ塩基対を用いたＰＣＲ産物の検出方法を示した模式図である。
［図３３］図３３は、Ｄｓｓ−ｈｘ−ｄＵアミダイト試薬の化学合成を示した結果である。
　条件：（ａ）Ｋ２ＣＯ３，ＤＭＦ；
５．　　（ｂ）Ｐｄ（ＰＰｈ３）４，ＣｕＩ，ＴＥＡ，ＤＭＦ；
６．　　（ｃ）　ＤＭＴｒＣｌ，ピリジン；
７．　　（ｄ）ＮＣ（ＣＨ２）２Ｏ−Ｐ（Ｃｌ）Ｎ（ｉＰｒ）２，ＤＩＥＡ，ＴＨＦ FIG. 1 shows an example of an artificial base pair of a quenching base (Pn, Px) and its complementary base (Ds or Dss). Examples are Pn and a fluorescent artificial base (Dss), and an artificial base pair consisting of Px and Ds.
FIG. 2 shows the structure of a quenching artificial base Pn and its 4 ′ derivative used in the examples of the present invention.
[FIG. 3] FIG. 3 shows the structures of the quenching artificial base Px and its derivatives used in Examples of the present invention.
FIG. 4 shows the structures of Ds and fluorescent artificial bases Dss, Dsss, and Dsav as examples of artificial bases complementary to Pn or Px used in the method of the present invention.
FIG. 5 shows the synthesis of Cy3-hx-dPxTP from NH 2 -hx-dPxTP. The reaction conditions are as follows. Among 100mM NaHCO 3, NH 2 -hx- dPxTP (8.4μmol), Na 2 CO 3 buffer (pH8.5) (500μl), in DMF (300μl), Cy3 (7.63μmol ) N- hydroxysuccinimide midges Luster, 12 hours at room temperature [FIG. 6] FIG. 6 shows the structure of a natural base amidite reagent bound with a fluorescent artificial base Dss or s.
The compounds in FIG. 6 are Dss-hx-dU amidite, s-hx-dU amidite, and s2-hx-dC amidite from the left.
FIG. 7 shows quenching of the fluorescent artificial base Dss in the complementary strand by the artificial base Pn. Each DNA solution (5 μM) in 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.1 mM EDAT was photographed by irradiation at 365 nm. When a single-stranded oligonucleotide (12-mer) containing a fluorescent artificial base (Dss) and a complementary oligonucleotide (12-mer) containing a quenching artificial base (Pn) are formed into a double strand, Dss Is quenched by Pn (FIG. 7, second from the left). Even when a base pair is formed with a natural base (such as T) or an artificial base (Dss or Ds), the fluorescence of Dss is not quenched (FIG. 7, third to fifth from the left).
FIG. 8 shows the fluorescence spectrum of each DNA fragment in the experiment of FIG. Double-stranded DNA (5′-GGTAACNATGCG-3 ′) containing single-stranded DNA containing Dss (5′-GGTAACDssATGCG-3 ′), Dss-Pn, Dss-Dss, Dss-Ds, and Dss-T base pairs. 8 is a result of fluorescence spectra (excitation wavelength: 385 nm, 25 ° C.) of a 5 μM DNA solution of (N = Dss) and 5′-CGCATN′GTTACC-3 ′ (N ′ = Dss, Ds, Pn or T). As shown in FIG. 5, the fluorescence of Dss was quenched by about one fifth by Pn.
FIG. 9 shows the results of examining the quenching action of Pn. Specifically, quenching in an aqueous solution of a 2′-deoxyribonucleoside 5′-triphosphate derivative (dDssTP) of the fluorescent artificial base Dss was examined as a concentration dependency of dPnTP. A: Changes in the fluorescence intensity of deoxyribonucleoside triphosphate (dDssTP, 5 μM) of the fluorescent artificial base Dss depending on the concentration dependence of deoxyribonucleoside triphosphate (dPnTP) of Pn. B: The result of comparing the quenching properties for dss of dPnTP and triphosphate of the natural type is shown. Steady-state Stern-Volmer plot of quenching of the fluorescent base dDssTP (5 μM) by Pn and the natural base deoxyribonucleoside triphosphate. Fluorescence due to excitation at 370 nm was measured at 20 ° C. in a solution of 100 mM NaCl, 10 mM sodium phosphate (pH 7.0), 0.1 mM EDTA, and a Stern-Volmer constant (K sv ) was calculated from the following equation.
Stern-Volmer equation: F 0 / F 1 = 1 + K SV [Q]
[F 0 and F 1 ,: fluorescence intensity with (F 1 ) with or without (F 0 ) quencher, respectively [Q]: concentration of quencher] Guanine bases are known to have quenching properties. However, Pn shows a stronger quenching property (FIG. 9B).
FIG. 10 shows the quenching characteristics of Ps and its various derivatives (FIGS. 2 and 3) and the fluorescence of Dss by Px. Specifically, fluorescence by excitation at 385 nm was measured at 25 ° C. in ethanol, and dDss (5 μM) in the presence of various Pn derivatives and Px deoxyribonucleoside (A: 2.5 mM, B: 5 mM). Changes in fluorescence intensity were examined. All of the derivatives showed stronger quenching characteristics than Pn.
FIG. 11 shows the results of examining a primer extension reaction using a template DNA containing Pn and dDssTP by Klenow fragment of DNA polymerase I derived from E. coli. Each concentration of dDssTP was added to 200 nM template DNA, 10 μM dCTP and dTTP, 0.1 U / μl Klenow fragment, reacted at 37 ° C. for 3 minutes, and analyzed by denaturing gel electrophoresis. Since dATP and dGTP are not added, the product when the extension reaction proceeds becomes a 33-mer that stops before C of the template. FIG. 11 shows that dDssTP is complementary to Pn in the template and incorporated into the complementary strand DNA. Although it has already been clarified that dDssTP is incorporated into Pa in the template (J. Am. Chem. Soc., 132: 4988-4989, 2010), Pn has higher Ds incorporation efficiency. I understood that. Further, when the concentration of dDssTP was increased, the extension reaction after Dss was incorporated into Pn and Pa was inhibited, but the primer extension reaction proceeded efficiently by decreasing the concentration of dDssTP.
FIG. 12 shows the results of examining PCR amplification of DNA containing Ds using Dss-Px base pairs. Ds-containing DNA (55-mer) was subjected to 20 cycles of PCR amplification using dDssTP, NH 2 -hx-dPxTP, and a natural base substrate. After denaturing gel electrophoresis, the product was stained with SYBR Green II. Was analyzed. It is the result of performing PCR by using template DNA (55-mer, S2) containing Ds and adding dDssTP and NH 2 -hx-dPxTP to a substrate (dNTPs) of a natural base. It was found that template DNA-S2 containing an artificial base was amplified as well as a template DNA containing only natural bases. Dss-Pn and Dss-Px base pairs function efficiently in PCR.
FIG. 13 shows the results of DNA sequencing after PCR amplification using Dss-Px base pairs. DNA (55-mer) containing Ds was subjected to PCR amplification for 15 cycles using dDssTP, NH 2 -hx-dPxTP, and a natural base substrate, and the amplification product was sequenced by a conventional method. It was found that 99% or more of Dss and NH 2 -hx-Px were retained in the amplified DNA. This sequencing uses the method developed by the inventors of the present application (Anna Unusual hydrophobic base pair system: site-specific incorporation of nucleic acids into RNA. , T. Fujiwara, R. Kawai, A. Sato, Y. Harada, S. Yokoyama, Nature Methods, 3, 729-735 (2006), An Unnatural base pir system. Kimot , R.Kawai, T.Mitsui, S.Yokoyama, and I.Hirao, Nucleic Acids Res., 37, e14 (2009))
FIG. 14 is a schematic diagram showing the principle of real-time PCR using Dss-Px base pairs. When Dss is introduced into a PCR primer and PCR is performed using dPnTP or dPxTP, Pn and Px are incorporated into the complementary strand in a complementary manner to Dss, and the fluorescence of Dss is quenched. By detecting this, real-time PCR becomes possible.
FIG. 15 shows the results of real-time PCR using Dss-Px base pairs. When the primer containing Dss shown in FIG. 14 is used, dPxTP is incorporated into its complementary strand, and the fluorescence of Dss is quenched. This forms a Dss-Px base pair during PCR, quenches the fluorescence of Dss, and enables application to real-time PCR.
Reaction mixture (25 μL scale)
1x Titanium Taq PCR buffer
1 μM 080731-5 ′ primer 3 (SEQ ID NO: 15)
1 μM 090914a-Plexor-Dss1 (SEQ ID NO: 16)
2 μM dPxTP
2 mM dNTPs
1 × Titanium TaqDNA polymerase
2aM (3 copies) -2fM (30000 copies) Template DNA application The total volume was adjusted to 25 μL with sterile water.
PCR conditions 94 ° C., 2 minutes → [94 ° C., 5 seconds-68 ° C., 40 seconds] × 55 cycles [FIG. 16] FIG. 16 shows the results of examining the fluorescence characteristics of DNA hairpins containing Dss-Pn base pairs. . The temperature dependency of the fluorescence intensity of each Dss of DNA hairpin (34-mer) containing Dss-Pn base pairs and single-stranded DNA (12-mer) containing Dss was measured. Each DNA was measured in a buffer solution containing 1 μM and 2 mM magnesium chloride. By introducing Dss and Pn into the stem region of the hairpin nucleic acid as a base pair, if the hairpin structure is formed, the fluorescence of Dss is quenched by Pn. When this hairpin DNA is thermally denatured, Strength increased. This Dss-Pn (or Dss-Px) base pair property can be applied to molecular beacons.
FIG. 17 shows that molecular beacons containing Dss-Pn base pairs can be visualized. The fluorescence of the hairpin beacon (26-mer) containing Dss-Pn base pairs was observed in the presence or absence of the target single-stranded DNA (71-mer). Each DNA was measured in 1 μM, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA. As a result, when the DNA complementary to the loop part of the molecular beacon containing Dss-Pn base pairs is recognized and a double strand is formed, the fluorescence quenching of Ds by Pn is eliminated, and the fluorescence of Dss is obtained by irradiating ultraviolet rays. It was confirmed with the naked eye.
[FIG. 18] FIG. 18 shows the result of detecting a single base mutation by a molecular beacon containing Dss-Pn base pairs. The fluorescence of each of the two types of hairpin type beacons (26-mer) containing Dss-Pn base pairs was observed in addition to the single-stranded DNA (71-mer) of each of the two types of target sequences with one base mutation. Each DNA was measured in 1 μM, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA. By creating a molecular beacon having a sequence complementary to each target DNA for two types of target sequences in the loop portion, it was possible to distinguish a difference of one base as a difference in emission intensity of Dss by hybridization.
FIG. 19 shows the principle of visualization PCR using Cy3-Px / Dss base pair as a substrate in which fluorescent dye Cy3 is bound to Px.
FIG. 20 shows an example of visual real-time PCR using Cy3-Px / Dss base pairs based on the principle described in FIG.
Reaction mixture (25 μL scale)
1x Titanium Taq PCR buffer
1 μM 080731-5 ′ primer 3 (SEQ ID NO: 15)
1 μM 090914a-Plexor-Dss1 (SEQ ID NO: 16)
2 μM Cy3-hx-dPxTP
2 mM dNTPs
1 × Titanium TaqDNA polymerase
2aM (3 copies) -200 fM (3000000 copies) Template DNA application The total volume was adjusted to 25 μL with sterile water.
PCR conditions 94 ° C., 2 minutes → [94 ° C., 5 seconds-68 ° C., 40 seconds] × 55 cycles Cy3 does not emit light at an excitation wavelength near 350 nm, and thus Px substrate bound to Cy3 (Cy3-hx-dPxTP) Does not emit light when irradiated with UV at 350 nm. When Cy3-hx-dPxTP is incorporated into the complementary strand of Dss due to base pairing of Dss-Px, fluorescence resonance energy transfer (FRET) from Dss to Cy3 occurs due to UV irradiation at 350 to 390 nm, and light is emitted. Thus, the DNA amplified by PCR can be detected with the naked eye by observing Cy3 emission through an orange filter.
FIG. 21 is a schematic diagram showing the principle of a real-time PCR method using a quenching Px base to which a fluorescent molecule (Cy3) is bound. When a fluorescent molecule (for example, Cy3) is bound to a quenching Px base, the fluorescence of the fluorescent molecule is quenched by about 30%. When this is used as a substrate (Cy3-hx-dPxTP) and PCR is performed using a primer into which a Ds base has been introduced, Cy3-hx-dPx is incorporated into DNA, thereby increasing the fluorescence intensity of Cy3. This method can be used for real-time PCR (FIG. 22).
FIG. 22 shows the results of real-time PCR using a quenching Px base to which a fluorescent molecule (Cy3) is bound. Real-time PCR detection using Cy3-hx-dPxTP as a substrate was performed using a real-time PCR apparatus (Stratagene, Mx3005P). PCR reaction was performed with primers: 1 μM each, natural base substrate dNTP: 0.2 mM each, artificial base substrate Cy3-hx-dPxTP: 2 μM, and a fluorescence change at 568 nm was detected at an excitation wavelength of 545 nm. Unlike the application example of FIG. 19 and FIG. 20, since Cy3 is directly excited by irradiation at 545 nm, the free substrate Cy3-hx-dPxTP also emits light. Therefore, this method cannot be identified with the naked eye as shown in FIG. Therefore, a real-time PCR apparatus was used to measure the increase in fluorescence intensity when Cy3-hx-dPxTP was incorporated into DNA.
FIG. 23 shows the result of gel electrophoresis detecting a real-time PCR product with a quenching Px base bound with a fluorescent molecule (Cy3). Since the PCR product shown in FIG. 22 incorporates Cy3, when this product is electrophoresed on an agarose gel, the PCR product is used on the gel without using a conventional dye for DNA staining such as EtBr or SYBR Green. Can be detected by Cy3 fluorescence.
Detection condition FLA7000 bioimaging analyzer (Cy3 mode)
532nm laser / O580 fluorescence filter PMT: 500V
[FIG. 24] FIG. 24 shows the results of examining the fluorescence characteristics of DNA containing a fluorescent molecule (Cy3) and a fluorescent artificial base s. Measurement conditions: 10 mM sodium phosphate (pH 7.0) containing 100 mM NaCl and 0.1 mM EDTA was prepared so that various DNA fragments were 5 μM, and luminescence was observed by irradiation at 254 to 365 nm. Lane 2 shows the case of DNA breakage containing one fluorescent artificial base s. When only s is introduced into DNA, s emission occurs by irradiation at 254 to 365 nm. However, when two adjacent s are introduced into DNA, the fluorescence is quenched (lane 3). When Cy3 is bound to a DNA fragment consisting only of a natural base, it does not emit light when irradiated with 365 nm (lane 4). However, when one or two s is introduced in the vicinity of Cy3 in the DNA, FRET occurs between s and Cy3, and Cy3 fluorescence is observed (lanes 5-7). Specifically, when s is excited at 365 nm, orange light emission of Cy3 is observed (lanes 5 and 6). Furthermore, when two s are introduced into the DNA fragment adjacent to each other, almost no light emission of s is observed even when excited at 254 to 365 nm by quenching of s (lane 3), but when Cy3 is bound to this DNA fragment, FRET occurs and Cy3 emission is observed (lane 7). Using this phenomenon, detection of DNA amplification by replication or transcription can be performed with the naked eye.
FIG. 25 shows the principle of a visualization PCR method in which a quenching Px base to which a fluorescent molecule (Cy3) is bound and a fluorescent artificial base s are combined. When two fluorescent artificial bases s are introduced adjacent to each other, the fluorescence of s is completely quenched and does not emit light when irradiated with 350 nm. Ds close to it does not emit light at 350 nm. When PCR is performed using two adjacent s and a primer containing Ds in the vicinity thereof and Cy3-hx-dPxTP, Cy3-hx-Px is incorporated into its complementary strand. Since two s and Ds are close to each other, FRET occurs by irradiation with 365 nm, which is the excitation wavelength of s, and a fluorescent dye such as Cy3 in the vicinity emits fluorescence (FIG. 26). Using this, DNA amplified by PCR can be detected with the naked eye (FIGS. 26 and 27).
[FIG. 26] FIG. 26 shows the results of a visualization PCR method in which a quenching Px base combined with a fluorescent molecule (Cy3) and a fluorescent artificial base s are combined. FIG. 26 shows the results of performing PCR, illuminating the PCR tube at 350 nm, and examining the light emission of Cy3 with the naked eye or through an orange filter. In the conventional method, it was difficult to confirm DNA amplification by PCR with the naked eye. That is, the conventional method using, for example, SYBR Green is the most used detection method in real-time PCR, but detection with the naked eye is difficult as shown on the right side in the figure. In contrast, the method of the present invention was able to detect PCR not only with real-time PCR but also with the naked eye (left side of FIG. 26).
[FIG. 27a] FIG. 27a shows the results of a visualization PCR method combining a quenching Px base to which a fluorescent molecule (Cy3) is bound and a fluorescent artificial base s. FIG. 28 shows the results of 55 cycles of PCR using 3 to 3 million copies of the target DNA and analyzing the PCR product by electrophoresis. In this detection system, the presence of Cy3 fluorescence by UV excitation at 365 nm can be observed at the reaction tube level without electrophoresis, and the amplification product can be detected with the naked eye even after 3 cycles of PCR after 55 cycles of PCR. It shows that it is possible.
[FIG. 27b] FIG. 27b shows the result of detecting a visualized PCR in which a quenching Px base bound with a fluorescent molecule (Cy3) and a fluorescent artificial base s are combined using a real-time quantitative PCR apparatus.
Reaction mixture (25μ scale)
1 μM 080731 5'primer 3
1 μM Primer 2d-Ds-ss3 3 ′ primer
2 μM Cy3-hx-dPxTP
200 μM dNTPs
1 x Titanium Taq Buffer
1 × Titanium Taq DNA Polymerase 2aM (3 copies) to 200 fM (3000000 copies) 98G template
PCR conditions: 94 ° C.-2 minutes → [94 ° C.-5 seconds → 68 ° C.-40 seconds] × 30-55 cycles This method is performed in real time due to the increase in Cy3 fluorescence intensity of Cy3-hx-Px incorporated into DNA. PCR is also possible.
FIG. 27c shows the result of visualizing the DNA amplification product of each PCR cycle of FIG. 27b.
FIG. 27d shows the result of quantifying the fluorescence intensity of each PCR tube in FIG. 27c. FIG. 27d1 is a plot of fluorescence intensity in each PCR cycle when 0, 3 to 30000 copies of DNA were amplified by PCR. FIG. 27d2 is a graph plotting the fluorescence intensity when 3-3,000,000 copies of each DNA were amplified in each PCR cycle.
[FIG. 28] FIG. 28 shows the detection of a product on gel electrophoresis by PCR (55 cycles) using a primer in which a quenching Px base bound to a fluorescent molecule (Cy3) and a fluorescent artificial base s are combined. The result of having performed is shown. By agarose gel electrophoresis of the visualized PCR product of FIG. 27a by the method of the present invention, the PCR product could be detected by irradiation at 312 nm or 532 nm. In the case of irradiation at 312 nm, FRET from s to Cy3 is detected, and in the case of irradiation at 532 nm, the result of direct excitation of Cy3 incorporated in DNA is shown. Since the PCR product is labeled with Cy3, it was possible to observe the PCR product on a gel by exciting s at 312 nm and FRET, or by directly exciting Cy3 at 532 nm.
[FIG. 29a] FIG. 29a uses a nucleoside derivative (FIG. 6, s-hx-dU, (Us)) in which a fluorescent molecule (s base) is bound to a natural base via a linker and a Ds-Px base pair. It is the schematic diagram which showed the detection method of the PCR product which was found. In the visualized PCR method of FIG. 25, two adjacent fluorescent artificial bases s are used. Instead, this fluorescent s is bound to a natural base via a linker, and this base is added to the PCR primer. A configuration in which two are adjacent to each other is the embodiment of FIG. 29a.
[FIG. 29b] FIG. 29b shows the use of a nucleoside derivative (FIG. 6, s-hx-dU, (Us)) obtained by binding a fluorescent molecule (s base) to a natural base via a linker and Ds-Px base pairing. It is the figure which showed each primer to be used, the arrangement | sequence of a template, and the conditions of PCR about PCR.
[FIG. 29c] FIG. 29c shows the results of visualization PCR in which a quenching Px base bound with a fluorescent molecule (Cy3) and a fluorescent artificial base s-hx-dU are combined. Real-time PCR is also possible by increasing the fluorescence intensity of Cy3 of Cy-hx-Px incorporated into DNA.
[FIG. 29d] FIG. 29d is a visualization of the DNA amplification products of each PCR cycle of FIG. 29c.
FIG. 30 is a chemical synthesis of s-hx-dU amidite reagent.
Conditions: (a) CBr 4 , PPh 3 , CH 2 Cl 2 ;
1. (B) K 2 CO 3 , DMF;
2. (C) Pac-Cl, HOBT, pyridine, CH 3 CN;
3. (D) DMTr-deoxy-5-iodouridine, Pd (PPh 3 ) 4 , CuI, TEA, DMF;
4). (E) NC (CH 2) 2 O-P (Cl) N (iPr) 2, DIEA, THF
FIG. 31 shows a nucleoside derivative (FIG. 6, s2-hx-dC, (Css)) in which two molecules of a fluorescent base (s) are bound to a natural base via a linker, and a Ds-Px base pair. It is the schematic diagram which showed the detection method of the PCR product using.
[FIG. 32] FIG. 32 shows a nucleotide derivative (FIG. 6, Dss-hx-dU (UDss)) in which a fluorescent molecule (Dss base) is bound to a natural base via a linker and a Ds-Px base pair. It is the schematic diagram which showed the detection method of PCR product.
FIG. 33 shows the results of chemical synthesis of Dss-hx-dU amidite reagent.
Conditions: (a) K 2 CO 3 , DMF;
5. (B) Pd (PPh 3 ) 4 , CuI, TEA, DMF;
6). (C) DMTrCl, pyridine;
7). (D) NC (CH 2) 2 O-P (Cl) N (iPr) 2, DIEA, THF
　本発明は、好ましくは以下の態様を含む。
　Ａ．蛍光性人工塩基と本発明の消光性人工性塩基の塩基対形成による蛍光の低下を利用する方法
　本発明の方法は、一態様において、蛍光性人工塩基と式ＩＩ The present invention preferably includes the following aspects.
A. Method of Utilizing Reduction of Fluorescence due to Base Pair Formation of Fluorescent Artificial Base and Quenching Artificial Base of the Present Invention In one aspect, the method of the present invention comprises a fluorescent artificial base and a formula II
Figure JPOXMLDOC01-appb-C000045
Figure JPOXMLDOC01-appb-C000045
　［式ＩＩにおいて、Ｒ２は、
　水素、水酸基、ＳＨ基、ハロゲン、
　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
　糖、糖鎖、アミノ酸、ペプチド、
　リンカーを介して結合した蛍光性分子、
からなる群から選択される基である］
で表される消光性人工塩基との塩基対形成により、蛍光性人工塩基の蛍光の低下を観察することにより、人工塩基対が形成されたことを検出する。 [In Formula II, R 2 is
Hydrogen, hydroxyl group, SH group, halogen,
A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
Sugar, sugar chain, amino acid, peptide,
A fluorescent molecule bound via a linker,
A group selected from the group consisting of]
The formation of an artificial base pair is detected by observing a decrease in fluorescence of the fluorescent artificial base by forming a base pair with the quenching artificial base represented by
　好ましくは、蛍光性人工塩基は式ＩＩと塩基対を形成することが知られている以下の、
　（ｉ）７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）；
　（ｉｉ）７−（２，２’，５’，２’’−ターチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓｓ）；
　（ｉｉｉ）２−アミノ−６−（２，２’−ビチエン−５−イル）プリン−９−イル基（ｓｓ）；
　（ｉｖ）２−アミノ−６−（２，２’，５’，２’’−ターチエン−５−イル）プリン−９−イル基（ｓｓｓ）；
　（ｖ）４−（２，２’−ビチエン−５−イル）−ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｓ）；
　（ｖｉ）４−［２−（２−チアゾリル）チエン−５−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｖ）；及び
　（ｖｉｉ）４−［５−（２−チエニル）チアゾール−２−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｖａｓ）；
からなる群より選択される。 Preferably, the fluorescent artificial base is known to form base pairs with Formula II:
(I) 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss);
(Ii) 7- (2,2 ′, 5 ′, 2 ″ -tertien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dsss);
(Iii) 2-amino-6- (2,2′-bithien-5-yl) purin-9-yl group (ss);
(Iv) 2-amino-6- (2,2 ′, 5 ′, 2 ″ -tert-en-5-yl) purin-9-yl group (sss);
(V) 4- (2,2′-bithien-5-yl) -pyrrolo [2,3-b] pyridin-1-yl group (Dsas);
(Vi) 4- [2- (2-thiazolyl) thien-5-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dsav); and (vii) 4- [5- (2-thienyl) ) Thiazol-2-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dvas);
Selected from the group consisting of
　その他にも蛍光性人工塩基として、２−アミノプリン、エテノアデノシンなどが利用されうる。
　好ましくは、本発明の消光性塩基は以下の式ＩＩ１又は式ＩＶである。 In addition, 2-aminopurine, etenoadenosine, and the like can be used as fluorescent artificial bases.
Preferably, the quenching base of the present invention is of formula II1 or formula IV below.
Figure JPOXMLDOC01-appb-C000046
Figure JPOXMLDOC01-appb-C000046
　［式ＩＩＩにおいて、Ｒ３は、
−Ｈ、ヨード、−ＣＨ３、 [In Formula III, R 3 is
-H, iodine, -CH 3,
Figure JPOXMLDOC01-appb-C000047
Figure JPOXMLDOC01-appb-C000047
　から選択される］ Selected from]
Figure JPOXMLDOC01-appb-C000048
Figure JPOXMLDOC01-appb-C000048
　［式ＩＶにおいて、Ｒ４は、
−ＣＨ３、−ＣＨ２−ＮＨ２、及び [In Formula IV, R 4 is
-CH 3, -CH 2 -NH 2 and,
Figure JPOXMLDOC01-appb-C000049
Figure JPOXMLDOC01-appb-C000049
　（ここにおいて、ｎは０ないし１２のいずれかの整数である）
から選択される］
　式ＩＶにおいて、ｎは好ましくは３−７、より好ましくは５である。 (Where n is an integer from 0 to 12)
Selected from]
In formula IV, n is preferably 3-7, more preferably 5.
　本発明はまた、
　７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）を塩基として有するポリヌクレオチドを含む、核酸プライマー；並びに、
　式ＩＩＩ又は式ＩＶの消光性塩基を塩基として有するポリヌクレオチドを含む、蛍光性人工塩基の蛍光の低下により、人工塩基の塩基対の形成を検出する方法に使用するためのキットを提供する。 The present invention also provides
A nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base; and
Provided is a kit for use in a method for detecting the formation of a base pair of an artificial base by reducing the fluorescence of the fluorescent artificial base, comprising a polynucleotide having a quenching base of formula III or formula IV as a base.
　Ｂ．本発明の消光性人工性塩基に結合した蛍光性分子の蛍光強度が人工塩基対の形成により変化することを利用する方法
　本発明は、一態様において、人工塩基対の形成を検出する方法であって、
　式Ｖ B. A method that utilizes the fact that the fluorescence intensity of a fluorescent molecule bound to the quenching artificial base of the present invention changes due to the formation of an artificial base pair The present invention, in one aspect, is a method for detecting the formation of an artificial base pair. And
Formula V
Figure JPOXMLDOC01-appb-C000050
Figure JPOXMLDOC01-appb-C000050
　［式Ｖにおいて、Ｒ５は、リンカーを介して結合した蛍光性分子である］
で表される消光性人工塩基中の蛍光性分子の蛍光強度が、式Ｖの人工塩基が塩基対を形成することによって変化し、人工塩基対が形成されたことを検出する、前記方法を提供する。 [In Formula V, R 5 is a fluorescent molecule bound via a linker]
Provided the method, wherein the fluorescence intensity of the fluorescent molecule in the quenching artificial base represented by the formula is detected by the fact that the artificial base of Formula V is changed by forming a base pair, and the artificial base pair is formed. To do.
　式Ｖの人工塩基が塩基対を形成する相補性の塩基対は、上述したＤｓ、Ｄｓｓ、Ｄｓｓｓ、ｓ、ｓｓ、ｓｓｓ、ｄＤｓａ、Ｄｓａｓ、Ｄｓａｖ、ｄＤｖａ、Ｄｖａｓ、及び、ｄＤｉａなど任意のものが可能である。好ましくは、Ｄｓ、ｓ、ｓｓ、ｓｓｓ、ｄＤｓａ、ｄＤｖａ、及び、ｄＤｉａ、より好ましくは７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）である。 Complementary base pairs in which the artificial base of Formula V forms a base pair include any of the above-described Ds, Dss, Dsss, s, ss, sss, dDsa, Dsas, Dsav, dDva, Dvas, and dDia. Is possible. Preferred are Ds, s, ss, sss, dDsa, dDva, and dDia, more preferably 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds).
　消光性人工塩基は、好ましくは、以下の式ＶＩの塩基： The quenching artificial base is preferably a base of the following formula VI:
Figure JPOXMLDOC01-appb-C000051
Figure JPOXMLDOC01-appb-C000051
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］である。
　リンカーは、式Ｉの消光剤について上述したものと同様のものを使用可能である。 [In Formula VI, R 6 is a fluorescent molecule attached via or without a linker].
Linkers similar to those described above for the quencher of formula I can be used.
　蛍光性分子は、式Ｉの消光剤について上述したものと同様のものを使用可能である。
　本発明はまた、Ｄｓ
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）を塩基とし
て有するポリヌクレオシドを含む、核酸プライマー、並びに、
　式ＶＩの塩基を有するポリヌクレオチド
を含む、蛍光強度の変化により、人工塩基の塩基対の形成を検出する方法に使用するためのキットを提供する。 Fluorescent molecules can be used similar to those described above for the quencher of formula I.
The present invention also provides Ds
A nucleic acid primer comprising a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base; and
Provided is a kit for use in a method for detecting the formation of an artificial base base pair by a change in fluorescence intensity, comprising a polynucleotide having a base of formula VI.
　Ｃ　蛍光共鳴エネルギー転移（ＦＲＥＴ）ならびに静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドを含む核酸を利用して核酸を検出する方法
　本発明は、一態様において、
　人工塩基対の形成を検出する方法であって、
　蛍光共鳴エネルギー転移（ＦＲＥＴ）ならびに静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドを含む核酸を利用し、当該核酸中の人工塩基（第一人工塩基）と蛍光性分子を有する人工塩基（第二人工塩基）との間で人工塩基対が形成されると、前記天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドから、第二人工塩基の有する蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などが生じて、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、前記方法、を提供する。 C. Utilizing a nucleic acid comprising a natural base modifier having a self-quenching property, an artificial base, or a polynucleoside having a base analog, which can serve as a donor, such as fluorescence resonance energy transfer (FRET) and static quenching (static quenching) In one aspect, the present invention provides a method for detecting a nucleic acid .
A method for detecting the formation of an artificial base pair,
Utilizing a nucleic acid comprising a natural base modifier having a self-quenching property, an artificial base, or a polynucleoside having a base analog, which can be a donor such as fluorescence resonance energy transfer (FRET) and static quenching, When an artificial base pair is formed between an artificial base (first artificial base) in the nucleic acid and an artificial base having a fluorescent molecule (second artificial base), the modified natural base, artificial base, or base Fluorescence resonance energy transfer or static quenching from a polynucleoside having an analog to a fluorescent molecule possessed by a second artificial base, resulting in a change in the fluorescence spectrum and formation of an artificial base pair Wherein the method is detected.
　核酸中の人工塩基（第一人工塩基）と蛍光性分子を有する人工塩基（第二人工塩基）との間で人工塩基対は、好ましくは、本発明の式ＩＩで表される消光性人工塩基を第二人工塩基として含む。しかしながら、必ずしもこれに限定されず、公知の人工塩基対について、蛍光共鳴エネルギー転移（ＦＲＥＴ）及び／又は静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などの供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドを含む核酸を利用することが可能である。 The artificial base pair between an artificial base (first artificial base) in a nucleic acid and an artificial base having a fluorescent molecule (second artificial base) is preferably a quenching artificial base represented by the formula II of the present invention As the second artificial base. However, the present invention is not necessarily limited thereto, and a known artificial base pair is a natural base modification having a self-quenching property that can be a donor such as fluorescence resonance energy transfer (FRET) and / or static quenching, It is possible to use a nucleic acid containing a polynucleoside having an artificial base or a base analog.
　Ｃ−１
　本発明は、Ｃの方法の一態様として以下の態様を提供する。
　本発明の蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法は、７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）と、以下の式ＶＩの塩基： C-1
The present invention provides the following aspects as one aspect of the method of C.
The method for detecting the formation of a base pair of an artificial base based on the change in the fluorescence spectrum due to the fluorescence resonance energy transfer or static quenching of the present invention is described in 7- (2,2′-biten-5-yl. ) Imidazo [4,5-b] pyridin-3-yl group (Dss) and a base of the following formula VI:
Figure JPOXMLDOC01-appb-C000052
Figure JPOXMLDOC01-appb-C000052
　［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
　２４０−４１０ｎｍの紫外線による励起により、Ｄｓｓから式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などが生じて、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、前記方法である。 In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
Excitation by 240-410 nm ultraviolet light causes fluorescence resonance energy transfer, static quenching, etc. from Dss to the fluorescent molecule in the base of formula VI, and the fluorescence spectrum changes, artificial base pairing It is the said method that it is detected that formed.
　本態様の模式図を図１９に示す。
　２４０−４１０ｎｍの紫外線はＤｓｓが励起される波長である。式ＶＩの塩基中の蛍光性分子は通常、この波長では蛍光を生じずＦＲＥＴは生じて始めて蛍光が観察されることが望ましい。 A schematic diagram of this embodiment is shown in FIG.
240-410 nm ultraviolet light is the wavelength at which Dss is excited. It is desirable that the fluorescent molecules in the base of formula VI normally do not fluoresce at this wavelength and that fluorescence is only observed when FRET occurs.
　以下のＣ−２ないしＣ−４の態様はいずれも７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、式ＶＩの塩基との間で人工塩基対の形成を検出するものである。 In any of the following C-2 to C-4 embodiments, an artificial base pair is formed between the 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and the base of formula VI. Is detected.
　Ｃ−２
　本発明は、Ｃの方法の一態様として以下の態様を提供する。
　本発明の蛍光共鳴エネルギー転移ならびに静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法は、
　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、式ＶＩの塩基との間で塩基対が形成されると、
　２４０−３９０ｎｍの紫外線による励起により、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などが生じ、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）を塩基として有するポリヌクレオチドが少なくとも１つ存在する、前記方法である。 C-2
The present invention provides the following aspects as one aspect of the method of C.
The method of detecting the base pair formation of an artificial base by the change of the fluorescence spectrum by the fluorescence resonance energy transfer and static quenching of the present invention,
When a base pair is formed between a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI,
Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in the base of formula VI by excitation with ultraviolet light at 240-390 nm Static quenching (static quenching) occurs, the fluorescence spectrum is changed, and it is detected that an artificial base pair is formed.
Here, at least one polynucleotide having a 2-amino-6- (2-thienyl) purin-9-yl group (s) as a base is present on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. This is the method.
　本態様の模式図を図２５に示す。
　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上存在するｓの数は限定されないが、好ましくは１−３、より好ましくは１又は２である。最も好ましくは２である。図２４、レーン３で示したようにｓが２個の場合には、ｓの自己消光性によりｓの蛍光強度が低下あるいは消光されており（自家消光）、ＦＲＥＴによる蛍光スペクトルの変化がよりはっきりと観察される（図２４、レーン７）。ｓが１個の場合はｓの蛍光が観察される（図２４、レーン２）。この場合も、ＦＲＥＴによりｓの蛍光から蛍光性分子の蛍光が観察されるようになる（図２４、レーン５、６）。 A schematic diagram of this embodiment is shown in FIG.
Here, the number of s present on the same strand as the nucleic acid containing a polynucleoside having Ds as a base is not limited, but is preferably 1-3, more preferably 1 or 2. Most preferably 2. As shown in FIG. 24, lane 3, when the number of s is 2, the fluorescence intensity of s is reduced or quenched due to the self-quenching property of s (self-quenching), and the change in the fluorescence spectrum due to FRET is clearer. (Fig. 24, lane 7). When s is 1, the fluorescence of s is observed (FIG. 24, lane 2). Also in this case, the fluorescence of the fluorescent molecule is observed from the fluorescence of s by FRET (FIG. 24, lanes 5 and 6).
　ｓが２つ並んだ場合のように２以上の人工塩基が同一核酸上に２つ以上存在している態様だけでなく、天然塩基に自己消光性塩基が結合している場合、Ｄｓｓのように１つの人工塩基中に２以上の消光性塩基部分（ｓ）が存在するような場合も本発明のＦＲＥＴ及び又は静的消光作用を利用した方法が適用可能である。 Not only in the case where two or more artificial bases are present on the same nucleic acid as in the case where two s are arranged, but when a self-quenching base is bound to a natural base, as in Dss Even when two or more quenching base moieties (s) are present in one artificial base, the method using the FRET and / or static quenching action of the present invention is applicable.
　Ｃ−３
　本発明は、Ｃの方法の一態様として以下の態様を提供する。
　本発明の蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法は、
　Ｄｓと、式ＶＩ塩基との間で塩基対が形成されると、
　３５０−３９０ｎｍの紫外線による励起により、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などが生じて蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）が天然型塩基に結合した少なくとも１の塩基を有するポリヌクレオチドが少なくとも１つ存在する、前記方法である。 C-3
The present invention provides the following aspects as one aspect of the method of C.
The method for detecting the base pair formation of an artificial base by the change of the fluorescence spectrum by the fluorescence resonance energy transfer or static quenching of the present invention,
When a base pair is formed between Ds and the Formula VI base,
Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI upon excitation with UV light at 350-390 nm Static quenching (static quenching) or the like occurs, and the fluorescence spectrum changes, and it is detected that an artificial base pair is formed.
Here, at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) is bonded to a natural base on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. Said method wherein there is at least one polynucleotide having one base.
　本態様の模式図を図２９ａ及び図３１に示す。
　ｓが結合する天然塩基の種類は限定されず、Ａ、Ｔ、Ｇ、Ｃ、Ｕのいずれでも可能である。また、ｓが結合する天然塩基が２以上隣接して存在する場合、隣接天然塩基は同じであっても、異なっていてもよい。好ましくは同じものが２以上隣接する。ｓが結合する天然塩基が同一核酸中に隣接する数も、ｓが核酸中に存在するＣ−２の態様と同様に、特に限定されないが、好ましくは１−３、より好ましくは１又は２である。最も好ましくは２である。 A schematic diagram of this embodiment is shown in FIGS. 29a and 31. FIG.
The type of natural base to which s binds is not limited, and any of A, T, G, C, and U is possible. When two or more natural bases to which s is bonded are present adjacent to each other, the adjacent natural bases may be the same or different. Preferably two or more of the same are adjacent. The number of natural bases to which s binds is adjacent to the same nucleic acid as well as the C-2 embodiment in which s is present in the nucleic acid, but it is preferably 1-3, more preferably 1 or 2. is there. Most preferably 2.
　また、Ｃ−３の態様には、１つの天然塩基に２以上のｓが結合する態様も含む（図３１）。結合するｓの数は、特に限定されないが好ましくは２又は３，より好ましくは２である。 In addition, the mode of C-3 includes a mode in which two or more s are bonded to one natural base (FIG. 31). The number of s to be bonded is not particularly limited, but is preferably 2 or 3, more preferably 2.
　Ｃ−４
　本発明は、Ｃの方法の一態様として以下の態様を提供する。
　本発明の蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法は、
　Ｄｓと、式ＶＩの塩基との間で塩基対が形成されると、
　２４０−４１０ｎｍの紫外線による励起により、７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移や静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などが生じて蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、少なくとも１つの７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドが存在する、
前記方法、である。 C-4
The present invention provides the following aspects as one aspect of the method of C.
The method for detecting the base pair formation of an artificial base by the change of the fluorescence spectrum by the fluorescence resonance energy transfer or static quenching of the present invention,
When a base pair is formed between Ds and the base of formula VI,
Fluorescence in the base of formula VI from 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) upon excitation with 240-410 nm UV light It is detected that the fluorescence spectrum changes due to fluorescence resonance energy transfer or static quenching to the molecule, and an artificial base pair is formed.
Here, at least one 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group on the same strand as the nucleic acid containing a polynucleoside having Ds as a base There is a polynucleotide having a base in which (Dss) is bound to a natural base,
Said method.
　本態様の模式図を図３２に示す。
　本発明のＣ−１ないしＣ−４を含むＣの態様において、蛍光性分子は特に限定されない。好ましくは、式Ｉの消光剤について上述したのと同様である。より好ましくはインドカルボシアニン（Ｃｙ３）である。 A schematic diagram of this embodiment is shown in FIG.
In the embodiment of C including C-1 to C-4 of the present invention, the fluorescent molecule is not particularly limited. Preferably, as described above for the quencher of formula I. More preferred is indocarbocyanine (Cy3).
　式ＶＩの塩基中の置換基Ｒ６は好ましくは以下の： The substituent R 6 in the base of formula VI is preferably:
Figure JPOXMLDOC01-appb-C000053
Figure JPOXMLDOC01-appb-C000053
　の構造を有する。
　本発明はまた、
　以下のｉ）−ｉｉｉ）からなる群から選択される、１つの核酸プライマー；
　ｉ）Ｄｓｓを塩基として有するポリヌクレオチドを含む、核酸プライマー；
　ｉｉ）Ｄｓを塩基として有するポリヌクレオシド、及び、少なくとも１つのｓを塩基として有するポリヌクレオチドを含む、核酸プライマー；
　ｉｉｉ）Ｄｓを塩基として有するポリヌクレオシド、及び、少なくとも１つのｓが天然型塩基に結合した少なくとも１の塩基を有するポリヌクレオチドを含む、核酸プライマー；及び
　ｉｖ）Ｄｓを塩基として有するポリヌクレオシド、及び、Ｄｓｓが天然型塩基に結合した塩基を有するポリヌクレオチドを含む、核酸プライマー、
　並びに、式ＶＩの塩基
を塩基として有するポリヌクレオチド
を含む、蛍光共鳴エネルギー転移ならびに静的消光作用（ｓｔａｔｉｃ　ｑｕｅｎｃｈｉｎｇ）などによる蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法に使用するためのキットを提供する。 It has the following structure.
The present invention also provides
One nucleic acid primer selected from the group consisting of i) -iii) below:
i) a nucleic acid primer comprising a polynucleotide having Dss as a base;
ii) a nucleic acid primer comprising a polynucleoside having Ds as a base and a polynucleotide having at least one s as a base;
iii) a nucleic acid primer comprising a polynucleoside having Ds as a base, and a polynucleotide having at least one s bonded to a natural base, and iv) a polynucleoside having Ds as a base; and A nucleic acid primer comprising a polynucleotide having a base in which Dss is bound to a natural base;
And a method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum by fluorescence resonance energy transfer and static quenching, including a polynucleotide having a base of formula VI as a base Providing a kit for
　Ｄｓｓ−ＰｎならびにＤｓｓ−Ｐｘ塩基対は、ＰＣＲにおいても効率よく機能する。本発明においては、核酸の塩基対は、転写、逆転写、複製又は翻訳のいずれの工程で形成されるものであってもよい。 Dss-Pn and Dss-Px base pairs function efficiently in PCR. In the present invention, the base pair of the nucleic acid may be formed by any step of transcription, reverse transcription, replication or translation.
　本発明のＦＲＥＴ及び／又は静的消光作用を利用した検出方法において（態様Ｃ）、検出スペクトルの変化は、肉眼で判定できる、ことを特徴とする。本発明前は、可視で簡便に人工塩基対の形成、標的核酸の検出を行うことのできる方法は存在しなかった。本発明の検出方法を利用すると、リアルタイムＰＣＲも可視化できる。よって、複雑かつ高価なＰＣＲ装置が不要となった
　また、本発明の人工塩基対の検出方法を利用して核酸の増幅を行った場合、増幅された核酸をそのまま電気泳動し、簡便に検出することが可能である（図２３等）。さらに、電気泳動のバンドの濃さにより定量化も可能である。 In the detection method using FRET and / or static quenching action of the present invention (Aspect C), the change in the detection spectrum can be determined with the naked eye. Prior to the present invention, there was no visible and simple method for forming an artificial base pair and detecting a target nucleic acid. Real-time PCR can also be visualized using the detection method of the present invention. Therefore, a complicated and expensive PCR apparatus is no longer necessary. In addition, when nucleic acid amplification is performed using the method for detecting an artificial base pair of the present invention, the amplified nucleic acid is electrophoresed as it is and is easily detected. It is possible (FIG. 23 etc.). Furthermore, quantification is also possible by the intensity of the electrophoresis band.
　以下、実施例により本発明をさらに具体的に説明するが、これらは本発明の技術的範囲を限定するためのものではない。当業者は本明細書の記載に基づいて容易に本発明に修飾・変更を加えることができ、それらは本発明の技術的範囲に含まれる。 Hereinafter, the present invention will be described more specifically by way of examples. However, these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.
　実施例１　Ｃｙ３−ｈｘ−ｄＰｘＴＰの化学合成（図５）
１）試薬、溶媒等
　試薬及び溶媒は、標準的な供給業者から購入し、さらに精製することなく使用した。１Ｈ−ＮＭＲ（３００ＭＨｚ）および３１Ｐ−ＮＭＲ（１２１ＭＨｚ）スペクトルは、ＢＲＵＫＥＲ　ＡＶ３００核磁気共鳴スペクトロメーター上に記録した。合成したヌクレオシド５’−三リン酸は、Ｇｉｌｓｏｎ　ＨＰＬＣシステムで最終精製を行った。エレクトロスプレイ−イオン化マススペクトル（ＥＳＩ−ＭＳ）　は、Ｗａｔｅｒｓ　２６９０　ＬＣシステムを伴ったＷａｔｅｒｓ　ＺＭＤ　４０００マスシステム上に記録した。 Example 1 Chemical Synthesis of Cy3-hx-dPxTP (FIG. 5)
1) Reagents, solvents, etc. Reagents and solvents were purchased from standard suppliers and used without further purification. 1 H-NMR (300 MHz) and 31 P-NMR (121 MHz) spectra were recorded on a BRUKER AV300 nuclear magnetic resonance spectrometer. The synthesized nucleoside 5′-triphosphate was subjected to final purification using a Gilson HPLC system. Electrospray-ionization mass spectra (ESI-MS) were recorded on a Waters ZMD 4000 mass system with a Waters 2690 LC system.
　２）１−（２−デオキシ−β−Ｄ−リボフラノシル−４−［３−（Ｃｙ３−カルボキサミドヘキサナミド）−１−プロピニル］−２−ニトロピロール　５’−三リン酸　（Ｃｙ３−ｈｘ−ｄＰｘＴＰ）　の合成
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−［３−（６−アミノヘキサナミド）−１−プロピニル］−２−ニトロピロール　５’−三リン酸（ＮＨ２−ｈｘ−ｄＰｘＴＰ）（８．４μｍｏｌ）の１００ｍＭＭ　ＮａＨＣＯ３−Ｎａ２ＣＯ３バッファー溶液（ｐＨ８．６，５００μｌ）にＣｙ３　Ｎ−ヒドロキシスクシンイミジルエステル（Ｃｙ３−ＳＥ，６．０ｍｇ，７．６３μｍｏｌ）のＤＭＦ（３００μｌ）溶液を加えて室温で１２時間静置した。５０ｍＭ　ＴＥＡＡ（３．０ｍｌ）を反応溶液に加えてＤＥＡＥ　Ｓｅｐｈａｄｅｘ　Ａ−２５ならびにＨＰＬＣで精製してＣｙ３−ｈｘ−ｄＰｘＴＰ（２．７μｍｏｌ，３５％）を得た。 2) 1- (2-Deoxy-β-D-ribofuranosyl-4- [3- (Cy3-carboxamidohexanamido) -1-propynyl] -2-nitropyrrole 5′-triphosphate (Cy3-hx-dPxTP) ) 1- (2-deoxy-β-D-ribofuranosyl) -4- [3- (6-aminohexanamido) -1-propynyl] -2-nitropyrrole 5′-triphosphate (NH 2 − hx-dPxTP) (8.4 μmol) in 100 mM NaHCO 3 —Na 2 CO 3 buffer solution (pH 8.6, 500 μl) with Cy3 N-hydroxysuccinimidyl ester (Cy3-SE, 6.0 mg, 7.63 μmol). DMF (300 μl) solution was added and allowed to stand at room temperature for 12 hours, 50 mM TEAA (3.0 ml) was added to the reaction solution and DEAE Sephade was added. Purification by A-25 and HPLC to give the Cy3-hx-dPxTP (2.7μmol, 35%).
　３）　Ｃｙ３−ｈｘ−ｄＰｘＴＰの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，Ｄ２Ｏ）　δ　８．５５（ｔ，１Ｈ，Ｊ＝１３．６Ｈｚ），７．９０（ｔ，２Ｈ，Ｊ＝１．７Ｈｚ），７．８５（ｄｄ，２Ｈ，Ｊ＝１．２，８．４Ｈｚ），７．７８（ｄ，１Ｈ，Ｊ＝２．１Ｈｚ），７．３９（ｄｄ，２Ｈ，Ｊ＝１．９，８．５Ｈｚ），７．１９（ｄ，１Ｈ，Ｊ＝２．１Ｈｚ），６．６４（ｔ，１Ｈ，Ｊ＝５．９Ｈｚ），６．３９（ｄｄ，２Ｈ，Ｊ＝２．８，１３．５Ｈｚ），４．５９（ｍ，１Ｈ），４．２２−４．０８（ｍ，９Ｈ），３．２０（ｑ，３２Ｈ，Ｊ＝７．３Ｈｚ），３．０７（ｔ，２Ｈ，Ｊ＝６．５Ｈｚ），２．５９（ｄｔ，１Ｈ，Ｊ＝６．１，１３．３Ｈｚ），２．３８（ｄｔ，１Ｈ，Ｊ＝６．２，１３．８Ｈｚ），２．２７−２．１７（ｍ，２Ｈ），１．８６（ｍ，２Ｈ），１．７７（ｓ，１２Ｈ），１．６７−１．５４（ｍ，４Ｈ），１．４２−１．２５（ｍ，５６Ｈ）．
　３１Ｐ　ＮＭＲ（１２１ＭＨｚ，Ｄ２Ｏ）　δ　−８．６５（ｂｓ，１Ｐ），−１０．７２（ｄ，１Ｐ，Ｊ＝１９．７Ｈｚ），−２２．３２（ｔ，１Ｐ，Ｊ＝２０．４Ｈｚ）．
　ＭＳ（ＥＳＩ）　ｆｏｒ　Ｃ４９Ｈ６５Ｎ６Ｏ２２Ｐ３Ｓ２　計算値：１２４７．２８（Ｍ＋Ｈ）＋，　実測値：１２４７．４３（Ｍ＋Ｈ）＋，　計算値：　１２４５．２８（Ｍ−Ｈ）−，　実測値：１２４４．９１（Ｍ−Ｈ）−．
　実施例２　人工塩基Ｐｎによる相補鎖中の蛍光性人工塩基Ｄｓｓの消光（図７）
　蛍光性人工塩基Ｄｓｓを含むＤＮＡ断片　（１２−ｍｅｒ、５’−ＧＧＴＡＡＣＮ１ＡＴＧＣＧ−３’、Ｎ１＝Ｄｓｓ）（配列番号１）のみ、もしくは相補鎖のＤＮＡ断片（１２−ｍｅｒ、５’−ＣＧＣＡＴＮ２ＧＴＴＡＣＣ−３’、Ｎ２＝Ｐｎ、Ｄｓｓ、Ｄｓ、もしくはＴ）（配列番号２）と二本鎖を形成させた場合の蛍光変化を調べるために、１０ｍＭ　リン酸ナトリウム（ｐＨ７．０），１００ｍＭ　ＮａＣｌ，０．１ｍＭ　ＥＤＴＡ溶液中で一本鎖ＤＮＡ（ｓｓＤＮＡ）および二本鎖ＤＮＡ（ｄｓＤＮＡ）が５μＭとなるように溶液を調製し、アニーリング後、３６５ｎｍでのＵＶトランスイルミネーター照射により蛍光を撮影した結果を図７に示した。 3) Physical property value of Cy3-hx-dPxTP 1 H NMR (300 MHz, D 2 O) δ 8.55 (t, 1H, J = 13.6 Hz), 7.90 (t, 2H, J = 1.7 Hz) 7.85 (dd, 2H, J = 1.2, 8.4 Hz), 7.78 (d, 1H, J = 2.1 Hz), 7.39 (dd, 2H, J = 1.9, 8) .5Hz), 7.19 (d, 1H, J = 2.1 Hz), 6.64 (t, 1H, J = 5.9 Hz), 6.39 (dd, 2H, J = 2.8, 13. 5 Hz), 4.59 (m, 1H), 4.22-4.08 (m, 9H), 3.20 (q, 32H, J = 7.3 Hz), 3.07 (t, 2H, J = 6.5 Hz), 2.59 (dt, 1H, J = 6.1, 13.3 Hz), 2.38 (dt, 1H, J = 6.2, 13.8 Hz), 2.27-2.17. ( , 2H), 1.86 (m, 2H), 1.77 (s, 12H), 1.67-1.54 (m, 4H), 1.42-1.25 (m, 56H).
31 P NMR (121 MHz, D 2 O) δ −8.65 (bs, 1P), −10.72 (d, 1P, J = 19.7 Hz), −22.32 (t, 1P, J = 20. 4 Hz).
MS (ESI) for C 49 H 65 N 6 O 22 P 3 S 2 Calculated value: 1247.28 (M + H) + , found value: 1247.43 (M + H) + , calculated value: 1245.28 (M−H) − , Actual measurement value: 1244.91 (M−H) − .
Example 2 Quenching of the fluorescent artificial base Dss in the complementary strand by the artificial base Pn (FIG. 7)
Only a DNA fragment (12-mer, 5′-GGTAACN 1 ATGCG-3 ′, N 1 = Dss) (SEQ ID NO: 1) containing a fluorescent artificial base Dss or a DNA fragment of a complementary strand (12-mer, 5′- CGCATN 2 GTTACC-3 ′, N 2 = Pn, Dss, Ds, or T) (SEQ ID NO: 2) In order to examine the fluorescence change when forming a double strand, 10 mM sodium phosphate (pH 7.0) , 100 mM NaCl, 0.1 mM EDTA solution to prepare single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) at 5 μM, and after annealing, fluoresce by UV transilluminator irradiation at 365 nm The result of photographing is shown in FIG.
　実施例３　各ＤＮＡ断片の蛍光スペクトル（図８）
　図８に、ＥＴＣ−２７３Ｔ温度コントローラーを装備したＪＡＳＣＯ　ＦＰ−６５００スペクトロメーターを用いて測定した各ＤＮＡ断片の蛍光スペクトルを示した。Ｄｓｓを含む一本鎖ＤＮＡ断片（１２−ｍｅｒ、５’−ＧＧＴＡＡＣＮ１ＡＴＧＣＧ−３’、Ｎ１＝Ｄｓｓ）（配列番号１）及びその相補鎖（１２−ｍｅｒ、５’−ＣＧＣＡＴＮ２ＧＴＴＡＣＣ−３’、Ｎ２＝Ｐｎ、Ｄｓｓ、Ｄｓ、もしくはＴ）（配列番号２）を含む二本鎖ＤＮＡを、１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０）、１００ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＴＡ溶液中で５μＭとなるように調製し、アニーリング後、３８５ｎｍでの励起による発光スペクトルを２５℃で測定した。 Example 3 Fluorescence spectrum of each DNA fragment (FIG. 8)
FIG. 8 shows the fluorescence spectrum of each DNA fragment measured using a JASCO FP-6500 spectrometer equipped with an ETC-273T temperature controller. Single-stranded DNA fragment containing Dss (12-mer, 5′-GGTAACN 1 ATGCG-3 ′, N 1 = Dss) (SEQ ID NO: 1) and its complementary strand (12-mer, 5′-CGCATN 2 GTTACC-3 ', N 2 = Pn, Dss, Ds, or T) (SEQ ID NO: 2) containing 5 μM of double-stranded DNA in 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA solution. After annealing, an emission spectrum by excitation at 385 nm was measured at 25 ° C.
　比較として、Ｄｓを含む一本鎖ＤＮＡ断片（１２−ｍｅｒ、５’−ＧＧＴＡＡＣＮ１ＡＴＧＣＧ−３’、Ｎ１＝Ｄｓ、５μＭ）（配列番号３）を用いて、３１０ｎｍでの励起による発光スペクトルを２５℃で測定した。 As a comparison, using a single-stranded DNA fragment containing Ds (12-mer, 5′-GGTAACN 1 ATGCG-3 ′, N 1 = Ds, 5 μM) (SEQ ID NO: 3), an emission spectrum by excitation at 310 nm was obtained. Measured at 25 ° C.
　実施例４　Ｐｎの消光作用（図９）
　Ａ．Ｐｎのデオキシリボヌクレオシド三リン酸（ｄＰｎＴＰ）の濃度依存による蛍光性人工塩基Ｄｓｓのデオキシリボヌクレオシド三リン酸（ｄＤｓｓＴＰ，５μＭ）の蛍光強度の変化
　ＥＴＣ−２７３Ｔ温度コントローラーを装備したＪＡＳＣＯ　ＦＰ−６５００スペクトロメーターを用いて、２ｍＭ、１ｍＭ、０．５ｍＭ、０．２ｍＭ、０．１ｍＭ、０．０５ｍＭのデオキシリボヌクレオシド三リン酸（ｄＰｎＴＰ）を含む１０ｍＭ　リン酸ナトリウム（ｐＨ７．０），１００ｍＭ　ＮａＣｌ，０．１ｍＭ　ＥＤＴＡ溶液（１００μｌ）に、デオキシリボヌクレオシド三リン酸（ｄＤｓｓＴＰ、１０５μＭ）を５μｌ加えて、３７０ｎｍでの励起によるｄＤｓｓＴＰの発光スペクトルを２０℃で測定した。 Example 4 Quenching action of Pn (FIG. 9)
A. Changes in fluorescence intensity of deoxyribonucleoside triphosphate (dDssTP, 5 μM) of fluorescent artificial base Dss depending on concentration of deoxyribonucleoside triphosphate (dPnTP) of Pn JASCO FP-6500 spectrometer equipped with ETC-273T temperature controller 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.1 mM EDTA containing 2 mM, 1 mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.05 mM deoxyribonucleoside triphosphate (dPnTP) 5 μl of deoxyribonucleoside triphosphate (dDssTP, 105 μM) was added to the solution (100 μl), and the emission spectrum of dDssTP by excitation at 370 nm was measured at 20 ° C.
　同様に天然型塩基のデオキシリボヌクレオシド三リン酸存在下のｄＤｓｓＴＰの蛍光消光効果を調べるために、１５ｍＭ、１２ｍＭ、９ｍＭ、６ｍＭ、３ｍＭ、１ｍＭのデオキシリボアデノシン三リン酸（ｄＡＴＰ）、デオキシリボグアノシン三リン酸（ｄＧＴＰ）、デオキシリボチミジン三リン酸（ｄＴＴＰ）、デオキシリボシチジン三リン酸（ｄＣＴＰ）を含む１０ｍＭ　リン酸ナトリウム（ｐＨ７．０），１００ｍＭ　ＮａＣｌ，０．１ｍＭ　ＥＤＴＡ溶液（１００μｌ）に、デオキシリボヌクレオシド三リン酸（ｄＤｓｓＴＰ、１０５μＭ）を５μｌ加えて、３７０ｎｍでの励起によるｄＤｓｓＴＰの発光スペクトルを２０℃で測定した。 Similarly, in order to examine the fluorescence quenching effect of dDssTP in the presence of the natural base deoxyribonucleoside triphosphate, 15 mM, 12 mM, 9 mM, 6 mM, 3 mM, 1 mM deoxyriboadenosine triphosphate (dATP), deoxyriboguanosine triphosphate In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, 0.1 mM EDTA solution (100 μl) containing (dGTP), deoxyribothymidine triphosphate (dTTP), deoxyribocytidine triphosphate (dCTP), deoxyribonucleoside triphosphate 5 μl of acid (dDssTP, 105 μM) was added, and the emission spectrum of dDssTP by excitation at 370 nm was measured at 20 ° C.
　Ｂ．ｄＰｎＴＰと天然型塩基の三リン酸とのＤｓｓに対する消光性の比較
　Ｐｎならびに天然型塩基のデオキシリボヌクレオシド三リン酸による、蛍光性人工塩基のヌクレオシド三リン酸、ｄＤｓｓＴＰ（５μＭ）の消光性をＳｔｅａｄｙ−ｓｔａｔｅ　Ｓｔｅｒｎ−Ｖｏｌｍｅｒ　ｐｌｏｔにより解析した。 B. Comparison of quenching properties of dPnTP and natural base triphosphate against Dss The quenching properties of the fluorescent artificial base nucleoside triphosphate, dDssTP (5 μM) by Pn and the natural base deoxyribonucleoside triphosphate Analysis by state Stern-Volmer plot.
　具体的には、１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０），１００ｍＭ　ＮａＣｌ，０．１ｍＭ　ＥＤＴＡ溶液中、２０℃で測定した発光スペクトル　（３７０ｎｍ　励起）を用いて、系中に存在するクエンチャー（ｄＰｎＴＰ，ｄＡＴＰ，ｄＧＴＰ，ｄＣＴＰ，ｄＴＴＰ）の濃度に対する蛍光強度の減少を以下のＳｔｅｒｎ−Ｖｏｌｍｅｒ式に代入することにより、Ｓｔｅｒｎ−Ｖｏｌｍｅｒ定数（ＫＳＶ）を算出した。 Specifically, a quencher (excited at 370 nm) measured in a 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA solution at 20 ° C. (excitation at 370 nm) The Stern-Volmer constant (K SV ) was calculated by substituting the decrease in fluorescence intensity with respect to the concentration of dPnTP, dATP, dGTP, dCTP, dTTP into the following Stern-Volmer equation.
　Ｓｔｅｒｎ−Ｖｏｌｍｅｒ式：　Ｆ０／Ｆ１＝１＋ＫＳＶ［Ｑ］
　ここで、Ｆ０は、クエンチャーの存在しない場合の蛍光強度、Ｆ１はクエンチャーの存在する場合の蛍光強度を示し、［Ｑ］はクエンチャーの濃度である。具体的には、クエンチャーの濃度［Ｑ］を横軸に、そのクエンチャー濃度に対するＦ０／Ｆ１を縦軸にプロットし、最小二乗法により得られた直線からＫＳＶを求めた。ここで、ＫＳＶの値が大きいほど消光能が強いクエンチャーであることを示す。グアニン塩基は消光性を有することが知られているが、Ｐｎはさらに強い消光性を示すことがわかった。 Stern-Volmer equation: F 0 / F 1 = 1 + K SV [Q]
Here, F 0 indicates the fluorescence intensity in the absence of the quencher, F 1 indicates the fluorescence intensity in the presence of the quencher, and [Q] is the quencher concentration. Specifically, the quencher concentration [Q] is plotted on the horizontal axis and F 0 / F 1 with respect to the quencher concentration is plotted on the vertical axis, and K SV was obtained from a straight line obtained by the least square method. Here, the larger the value of K SV is, the stronger the quenching ability is. Although guanine base is known to have quenching properties, Pn has been found to exhibit even stronger quenching properties.
実施例５　ｄＰｎとその各種誘導体によるｄＤｓｓの蛍光の消光（図１０）
　５ｍＭまたは５ｍＭ　ｄＰｎまたはその各種誘導体存在下での終濃度５μＭ　ｄＤｓｓの蛍光を、励起波長３８５ｎｍ、測定温度２５℃で測定した結果を図１０に示した。具体的には、各ヌクレオシド溶液（２０μＭ　ｄＤｓｓ、２０ｍＭ　ｄＰｎおよびその各種誘導体）を以下の手順で調製した。 Example 5 Quenching of dDss fluorescence by dPn and its various derivatives (FIG. 10)
FIG. 10 shows the results of measuring fluorescence at a final concentration of 5 μM dDss in the presence of 5 mM or 5 mM dPn or various derivatives thereof at an excitation wavelength of 385 nm and a measurement temperature of 25 ° C. Specifically, each nucleoside solution (20 μM dDss, 20 mM dPn and various derivatives thereof) was prepared by the following procedure.
　ｄＤｓｓ、ｄＰｎまたはその各種誘導体をそれぞれ約５ｍｇとり、５５−６０℃で６時間乾燥後、秤量し、ｄＤｓｓは２ｍＭ、ｄＰｎまたはその各種誘導体はそれぞれ２０ｍＭになるように２０％　アセトニトリル水溶液を加えた。ｄＤｓｓはさらに希釈し、２０μＭとした。蛍光スペクトル測定用のサンプルに関しては、ｄＰｎまたはその各種誘導体の終濃度２．５ｍＭの場合（図１０Ａ）、５０μｌの２０μＭ　ｄＤｓｓ溶液、２５μｌの２０ｍＭ　ｄＰｎまたはその各種誘導体溶液、２５μｌの２０％アセトニトリル溶液、１００μｌのエタノールを混合して、全量を２００μｌとした。ｄＰｎまたはその各種誘導体の終濃度５ｍＭの場合（図１０Ｂ）には、５０μｌの２０μＭ　ｄＤｓｓ溶液、５０μｌの２０ｍＭ　ｄＰｎまたはその各種誘導体溶液、１００μｌのエタノールを混合して、全量を２００μｌとした。 About 5 mg each of dDss, dPn or various derivatives thereof was taken, dried at 55-60 ° C. for 6 hours, weighed, and 20% acetonitrile aqueous solution was added so that dDss was 2 mM and dPn or various derivatives thereof were 20 mM. dDss was further diluted to 20 μM. Regarding the sample for measuring the fluorescence spectrum, when the final concentration of dPn or its various derivatives is 2.5 mM (FIG. 10A), 50 μl of 20 μM dDss solution, 25 μl of 20 mM dPn or various derivatives thereof, 25 μl of 20% acetonitrile solution, 100 μl of ethanol was mixed to make a total volume of 200 μl. When the final concentration of dPn or its various derivatives was 5 mM (FIG. 10B), 50 μl of 20 μM dDss solution, 50 μl of 20 mM dPn or various derivatives thereof, and 100 μl of ethanol were mixed to make a total volume of 200 μl.
　実施例６　クレノウ断片によるＤｓｓ−Ｐｎ塩基対のＤＮＡ中への一塩基取り込み実験（表１）
　クレノウ断片による一塩基取り込み実験を、文献に従っておこなった（Ｋｉｍｏｔｏ，Ｍ．；Ｙｏｋｏｙａｍａ，Ｓ．；Ｈｉｒａｏ，Ｉ．Ｂｉｏｔｅｃｈｎｏｌ．Ｌｅｔｔ．２００４，２６，９９９−１００５、Ｐｅｔｒｕｓｋａ，Ｊ．；Ｇｏｏｄｍａｎ，Ｍ．Ｆ．；Ｂｏｏｓａｌｉｓ，Ｍ．Ｓ．；Ｓｏｗｅｒｓ，Ｌ．Ｃ．；Ｃｈｅｏｎｇ，Ｃ．；Ｔｉｎｏｃｏ，Ｉ．Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ　１９８８，８５，６２５２−６２５６、Ｇｏｏｄｍａｎ，Ｍ．Ｆ．；Ｃｒｅｉｇｈｔｏｎ，Ｓ．；Ｂｌｏｏｍ，Ｌ．Ｂ．；Ｐｅｔｒｕｓｋａ，Ｊ．Ｃｒｉｔ．Ｒｅｖ．Ｂｉｏｃｈｅｍ．Ｍｏｌ．Ｂｉｏｌ．１９９３，２８，８３−１２６、Ｍｏｒａｌｅｓ，Ｊ．Ｃ．；Ｋｏｏｌ，Ｅ．Ｔ．Ｎａｔ．Ｓｔｒｕｃｔ．Ｂｉｏｌ．１９９８，５，９５０−９５４）。 Example 6 Single base incorporation experiment into DNA of Dss-Pn base pair by Klenow fragment (Table 1)
Single base incorporation experiments with Klenow fragment were performed according to the literature (Kimoto, M .; Yokoyama, S .; Hirao, I. Biotechnol. Lett. 2004, 26, 999-1005, Petruska, J .; Goodman, M. F. Boosalis, MS; Sowers, LC; Cheong, C .; Tinoco, I. Proc. Natl. Acad. Sci. USA 1988, 85, 6252-6256, Goodman, MF; Bloom, LB; Petruska, J. Crit. Rev. Biochem. MoI. Biol., 1993, 28, 83-126, Morales, J. C., Kool, ET Nat. Biol.1 998, 5, 950-954).
　具体的には、５’末端が６−カルボキシフルオレセインで標識されたプライマー（２０−ｍｅｒ、５’−ＡＣＴＣＡＣＴＡＴＡＧＧＧＡＧＧＡＡＧＡ−３’（配列番号４）または、５’−ＡＣＴＣＡＣＴＡＴＡＧＧＧＡＧＣＴＴＣＴ−３’（配列番号５））と鋳型ＤＮＡ（３５−ｍｅｒ、５’−ＡＧＣＴＣＴＤｓｓＴＣＴＴＣＣＴＣＣＣＴＡＴＡＧＴＧＡＧＴＣＧＴＡＴＴＡＴ−３’（配列番号６））または、５’−ＴＣＧＡＧＡＮＡＧＡＡＧＣＴＣＣＣＴＡＴＡＧＴＧＡＧＴＣＧＴＡＴＴＡＴ−３’（Ｎ＝Ｐｎ、Ａ、Ｇ、Ｃ、Ｔ）（配列番号７））を、２０ｍＭ　ＭｇＣｌ２、２ｍＭ　ＤＴＴ、および１００μｇ／ｍｌウシ血清アルブミン（ＢＳＡ）を含有する１００ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）　緩衝液中で、９５℃で加温した後に、４℃へ緩やかに冷却することによりアニーリングし、鋳型鎖とプライマーの二本鎖を形成させた。 Specifically, a primer labeled with 6-carboxyfluorescein at the 5 ′ end (20-mer, 5′-ACTCACTATAGGGAGGAAGA-3 ′ (SEQ ID NO: 4) or 5′-ACTCACTATAGGGAGCTTCT-3 ′ (SEQ ID NO: 5)) And template DNA (35-mer, 5′-AGCTCTDsTCTCTCCCCCCTATAGTGAGTCCGTATTAT-3 ′ (SEQ ID NO: 6)) or 5′-TCGAGANAGAAGCCCCCTATAGTGAGTCGTATTAT-3 ′ (N = Pn, A, G, C, T) (SEQ ID NO: 7)) , 20mM MgCl 2, 2mM DTT, and in 100mM Tris-HCl (pH7.5) buffer containing 100 [mu] g / ml bovine serum albumin (BSA), after heated at 95 ° C., slow to 4 ° C. Annealed by crab cooling, to form a double strand of the template strand and the primer.
　このプライマー—鋳型二本鎖ＤＮＡ溶液（１０μＭ）を５μｌずつ分注した後，エキソヌクレアーゼ活性を持たないクレノウ断片（ＫＦｅｘｏ−、Ａｍｅｒｓｈａｍ　ＵＳＢ）の酵素溶液（２μｌ）を加えて、３７℃で２分間インキュベートし、ＤＮＡ・酵素複合体を形成させた。その溶液に３μｌの各基質、即ちヌクレオシド三リン酸溶液（Ｄｓｓ、ＰｎまたはＡ、Ｇ、Ｃ、Ｔのうち１種、１μＭ−５ｍＭ）を加えて、３７℃で酵素反応（１−３５分間）を行った。反応の終了は、１０μｌの２０ｍＭ　ＥＤＴＡを含む９５％ホルムアミド溶液（停止溶液）を加えて７５℃で３分加温することで行った。 After 5 μl of this primer-template double-stranded DNA solution (10 μM) was dispensed, an enzyme solution (2 μl) of Klenow fragment having no exonuclease activity (KFexo-, Amersham USB) was added, and then at 37 ° C. for 2 minutes. Incubation was performed to form a DNA / enzyme complex. 3 μl of each substrate, ie, nucleoside triphosphate solution (Dss, Pn or one of A, G, C, T, 1 μM-5 mM) was added to the solution, and the enzyme reaction was performed at 37 ° C. (1-35 minutes). Went. The reaction was completed by adding a 95% formamide solution (stop solution) containing 10 μl of 20 mM EDTA and heating at 75 ° C. for 3 minutes.
　反応条件をまとめると以下の通りである。溶液（１０μｌ）中、５μＭプライマー−鋳型二本鎖、５−５０ｎＭ酵素及び０．３−１５００μＭ基質を使用。溶液（１０μｌ）は、５０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）、１０ｍＭ　ＭｇＣｌ２、１ｍＭ　ＤＴＴ及び０．０５ｍｇ／ｍｌ　ＢＳＡを含む。反応は、３７℃で１−３５分間。 The reaction conditions are summarized as follows. Use 5 μM primer-template duplex, 5-50 nM enzyme and 0.3-1500 μM substrate in solution (10 μl). The solution (10 μl) contains 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 1 mM DTT and 0.05 mg / ml BSA. The reaction is 1-35 minutes at 37 ° C.
　反応溶液の一部を停止溶液で希釈した後、その希釈反応液０．５μｌをローディング溶液（脱イオンホルムアミド：２５ｍＭ　ＥＤＴＡを含む５０ｍｇ／ｍｌブルーデキストラン溶液＝５：１）３μｌと混合し、９０℃で２分加熱し、氷上に置いて急冷した。そのうちの約０．５μｌを１レーンおきにシークエンスゲルにロードし電気泳動を行った。シークエンスゲル（３６ｃｍ　ＷＴＲ）の組成は、６Ｍ　尿素、１０％ポリアクリルアミド（アクリルアミド：ビスアクリルアミド＝１９：１）、０．５×ＴＢＥである。泳動用緩衝液は，０．５×ＴＢＥを用いた。Ｒｕｎ　Ｍｏｄｕｌｅは、ＧＳ　Ｒｕｎ　３６Ｃ−２４００である。泳動時間は約１時間とし、反応産物のピークパターンの解析及び定量は、ＧｅｎｅＳｃａｎソフトウエア　（バージョン３．０）装備した自動ＡＢＩ３７７ＤＮＡシークエンサーで解析した。 After diluting a part of the reaction solution with the stop solution, 0.5 μl of the diluted reaction solution is mixed with 3 μl of the loading solution (deionized formamide: 50 mg / ml blue dextran solution containing 5 mM EDTA = 5: 1), and the temperature is 90 ° C. For 2 minutes and then placed on ice to quench. About 0.5 μl of this was loaded onto a sequence gel every other lane and subjected to electrophoresis. The composition of the sequence gel (36 cm WTR) is 6M urea, 10% polyacrylamide (acrylamide: bisacrylamide = 19: 1), 0.5 × TBE. 0.5 × TBE was used as the electrophoresis buffer. The Run Module is GS Run 36C-2400. The electrophoresis time was about 1 hour, and the peak pattern of the reaction product was analyzed and quantified with an automatic ABI377 DNA sequencer equipped with GeneScan software (version 3.0).
　未反応のプライマー断片，および一塩基とりこまれて伸長したＤＮＡ断片のピークの面積を用いて、一ヌクレオチド伸長されたプライマーの割合を定量し、Ｈａｎｅｓ−Ｗｏｏｌｆ　ｐｌｏｔ（Ｇｏｏｄｍａｎ，Ｍ．Ｆ．；Ｃｒｅｉｇｈｔｏｎ，Ｓ．；Ｂｌｏｏｍ，Ｌ．Ｂ．；Ｐｅｔｒｕｓｋａ，Ｊ．Ｃｒｉｔ．Ｒｅｖ．Ｂｉｏｃｈｅｍ．Ｍｏｌ．Ｂｉｏｌ．１９９３，２８，８３−１２６）により酵素学的パラメーターＶｍａｘ、ＫＭを算出した。なお、Ｖｍａｘの値は、使用された種々の酵素および二本鎖濃度に対して酵素濃度２０ｎＭ、二本鎖濃度５μＭに標準化された。 Using the peak areas of the unreacted primer fragment and the DNA fragment extended by incorporation of a single base, the proportion of the primer extended by one nucleotide was quantified, and Hanes-Woolp plot (Goodman, MF; Creighton, S.; Bloom, L.B;. Petruska , enzymatic parameters V max by J.Crit.Rev.Biochem.Mol.Biol.1993,28,83-126), were calculated K M. The value of V max, the enzyme concentration 20nM for the various enzymes and double strand concentrations used were normalized to the duplex concentration 5 [mu] M.
　結果を表１に示す Results are shown in Table 1.
Figure JPOXMLDOC01-appb-T000054
Figure JPOXMLDOC01-appb-T000054
　実施例７　大腸菌由来のＤＮＡポリメラーゼ　Ｉ　のクレノウ断片を用いたＰｎを含む鋳型ＤＮＡとｄＤｓｓＴＰによるプライマー伸長反応（図１１）
　５’末端を３２Ｐで標識したプライマー（２３−ｍｅｒ）（配列番号８）とＰｎもしくはＰａを含む鋳型ＤＮＡ（３５−ｍｅｒ）（配列番号９）を、１４ｍＭ　ＭｇＣｌ２、０．２ｍＭ　ＤＴＴを含有する２０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５）緩衝液中で、９５℃で加温後、４℃へ緩やかに冷却することによりアニーリングし、鋳型鎖とプライマーの二本鎖を形成させた。このプライマー—鋳型二本鎖ＤＮＡ溶液（４００ｎＭ）を５μｌずつ分注した後、２．５μｌの各基質、即ちヌクレオシド三リン酸溶液（４０μＭ　ｄＣＴＰ、４０μＭ　ｄＴＴＰ、０−４０μＭ　ｄＤｓｓＴＰ）を氷上で加えた。この溶液に、エキソヌクレアーゼ活性をもつクレノウ断片（ＫＦ　ｅｘｏ＋、ＴａＫａＲａ）を滅菌水で希釈した酵素溶液（２．５μｌ、１ユニット）を加えることで反応を開始し、３７℃で３分間保温後、１０μｌの１０Ｍ尿素を含む１×ＴＢＥ溶液（停止溶液）を加えて７５℃で３分加温することで反応を止めた。反応産物を１５％ポリアクリルアミド—７　Ｍ　尿素ゲルで電気泳動し、バンドパターンをバイオイメージングアナライザー（ＦＬＡ７０００、富士フィルム）によるオートラジグラフィーにより解析した。 Example 7 Primer extension reaction using Pn-containing template DNA and dDssTP using Klenow fragment of DNA polymerase I derived from E. coli (FIG. 11)
Primer (23-mer) (SEQ ID NO: 8) labeled with 32 P at the 5 ′ end and template DNA (35-mer) (SEQ ID NO: 9) containing Pn or Pa, containing 14 mM MgCl 2 and 0.2 mM DTT In 20 mM Tris-HCl (pH 7.5) buffer, the sample was heated at 95 ° C. and then slowly cooled to 4 ° C. to form a template strand and a primer duplex. After 5 μl of this primer-template double-stranded DNA solution (400 nM) was dispensed, 2.5 μl of each substrate, ie, nucleoside triphosphate solution (40 μM dCTP, 40 μM dTTP, 0-40 μM dDssTP) was added on ice. . The reaction was started by adding an enzyme solution (2.5 μl, 1 unit) obtained by diluting Klenow fragment having exonuclease activity (KF exo +, TaKaRa) with sterilized water to this solution. After incubating at 37 ° C. for 3 minutes, The reaction was stopped by adding 1 × TBE solution (stop solution) containing 10 μl of 10M urea and heating at 75 ° C. for 3 minutes. The reaction product was electrophoresed on a 15% polyacrylamide-7 M urea gel, and the band pattern was analyzed by autoradiography using a bioimaging analyzer (FLA7000, Fuji Film).
　実施例８　Ｄｓｓ−Ｐｘ塩基対を用いたＤｓを含むＤＮＡのＰＣＲ増幅（図１２）
　所定濃度の人工塩基基質、ＮＨ２−ｈｘ−ｄＰｘＴＰおよびｄＤｓｓＴＰ、の存在下で、Ｄｓを含む鋳型ＤＮＡ（Ｓ２、５５−ｍｅｒ）もしくは天然型塩基だけからなるＤＮＡ（対照、５５−ｍｅｒ）を用いてＰＣＲを行い、その産物を電気泳動により解析した結果を図１２に示した。 Example 8 PCR amplification of DNA containing Ds using Dss-Px base pairs (FIG. 12)
In the presence of a predetermined concentration of artificial base substrates, NH 2 -hx-dPxTP and dDssTP, template DNA containing Ds (S2, 55-mer) or DNA consisting only of natural bases (control, 55-mer) is used. PCR was performed, and the result of analyzing the product by electrophoresis is shown in FIG.
　使用した鋳型ＤＮＡおよびプライマーの配列は以下のとおりである。
　ＤＮＡ　Ｓ２　（５５−ｍｅｒ、下線部はプライマーのアニーリング部位）
　５’−ＴＴＴＣＡＣＡＣＡＧＧＡＡＡＣＡＧＣＴＡＴＧＡＣＧＧＣＣＣＤｓＴＴＧＣＣＣＴＡＴＡＧＴＧＡＧＴＣＧＴＡＴＴＡＴＣ−３’（配列番号１０）
　ＤＮＡ　対照　（５５−ｍｅｒ、下線部はプライマーのアニーリング部位）
　５’−ＴＴＴＣＡＣＡＣＡＧＧＡＡＡＣＡＧＣＴＡＴＧＡＣＧＧＡＴＣＣＡＴＴＣＣＣＴＡＴＡＧＴＧＡＧＴＣＧＴＡＴＴＡＴＣ−３’（配列番号１１）
　５’側プライマー：
　５’−ＣＧＴＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＧＡＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧ−３’（配列番号１２）
　３’側プライマー：
　５’−ＴＴＴＣＡＣＡＣＡＧＧＡＡＡＣＡＧＣＴＡＴＧＡＣ−３’（配列番号１３）
　ＰＣＲ（反応スケール：４０μｌ）は鋳型として最終濃度０．４ｎＭのＤＮＡ断片を用いて実施し、９４℃−３０秒、４５℃−３０秒、６５℃−４分を１サイクルとして２０サイクル行った。反応液の最終組成は２０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．８）、１０ｍＭ　ＫＣｌ、１０ｍＭ（ＮＨ４）２ＳＯ４、２ｍＭ　ＭｇＳＯ４、０．１％　ＴｒｉｔｏｎＸ−１００、ＤｅｅｐＶｅｎｔ　ＤＮＡポリメラーゼ（０．０２ユニット／μｌ、ＮＥＢ）、１μＭ　５’側プライマー、１μＭ　３’側プライマー、０．３ｍＭ　各天然型塩基基質ｄＮＴＰ、１０−２５μＭ　ｄＤｓｓＴＰ、および２５μＭ　ＮＨ２−ｈｘ−ｄＰｘＴＰである。２０サイクル後のＰＣＲ産物を１５％ポリアクリルアミド—７Ｍ　尿素ゲルで電気泳動した。 The template DNA and primer sequences used are as follows.
DNA S2 (55-mer, underlined primer annealing site)
5′- TTTCACACAGGAAAACAGCTATGAC GGCCCDsTTGCC CTATAGTGAGTCGTATTATC- 3 ′ (SEQ ID NO: 10)
DNA control (55-mer, underlined primer annealing site)
5′- TTTCACACAGGAAAACAGCTATGAC GGATCCATTCC CTATAGTGAGTCGTATTATTC- 3 ′ (SEQ ID NO: 11)
5 'primer:
5'-CGTTGTAAAACGACGGGCCAGGATAATACGACTCACTATAG-3 '(SEQ ID NO: 12)
3 'primer:
5′-TTTCACACAGGAAACAGCTATGAC-3 ′ (SEQ ID NO: 13)
PCR (reaction scale: 40 μl) was performed using a DNA fragment having a final concentration of 0.4 nM as a template, and 20 cycles of 94 ° C. for 30 seconds, 45 ° C. for 30 seconds, and 65 ° C. for 4 minutes were performed. The final composition of the reaction solution was 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, Deep Vent DNA polymerase (0.02 unit / μl). , NEB), 1 μM 5 ′ primer, 1 μM 3 ′ primer, 0.3 mM each natural base substrate dNTP, 10-25 μM dDssTP, and 25 μM NH 2 -hx-dPxTP. The PCR product after 20 cycles was electrophoresed on a 15% polyacrylamide-7M urea gel.
　増幅されたＤＮＡのバンドは、ゲルをＳＹＢＲ　Ｇｒｅｅｎ　ＩＩ　（Ｌｏｎｚａ）で染色した後、バイオイメージャーＬＡＳ４０００（富士フィルム）　のＳＹＢＲモードで検出した。 The amplified DNA band was detected in SYBR mode of Bioimager LAS4000 (Fuji Film) after the gel was stained with SYBR Green II (Lonza).
　実施例９　Ｄｓｓ−Ｐｘ塩基対を用いたＰＣＲ増幅後のＤＮＡのシーケンシング（図１３）
　所定濃度の人工塩基基質、ＮＨ２−ｈｘ−ｄＰｘＴＰおよびｄＤｓｓＴＰ、の存在下で、Ｄｓを含む鋳型ＤＮＡ（Ｓ２、５５−ｍｅｒ）を用いてＰＣＲを行い、その産物中に人工塩基Ｄｓｓが保持されているかを、人工塩基基質ｄＰａ’ＴＰもしくはｄｄＰａ’ＴＰを利用したＤＮＡシーケンシングにより解析した結果を図１３に示した。 Example 9 Sequencing of DNA after PCR amplification using Dss-Px base pairs (FIG. 13)
PCR is performed using a template DNA (S2, 55-mer) containing Ds in the presence of a predetermined concentration of artificial base substrates, NH 2 -hx-dPxTP and dDssTP, and the artificial base Dss is retained in the product. FIG. 13 shows the result of analysis by DNA sequencing using an artificial base substrate dPa′TP or ddPa′TP.
　使用した鋳型ＤＮＡおよびプライマーの配列は以下のとおりである。
　ＤＮＡ　Ｓ２　（５５−ｍｅｒ、下線部はプライマーのアニーリング部位）
　５’−ＴＴＴＣＡＣＡＣＡＧＧＡＡＡＣＡＧＣＴＡＴＧＡＣＧＧＣＣＣＤｓＴＴＧＣＣＣＴＡＴＡＧＴＧＡＧＴＣＧＴＡＴＴＡＴＣ−３’（配列番号１０）
　ＰＣＲ用プライマー
　５’側プライマー：５’−ＣＧＴＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＧＡＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧ−３’（配列番号１２）
　３’側プライマー：５’−ＴＴＴＣＡＣＡＣＡＧＧＡＡＡＣＡＧＣＴＡＴＧＡＣ−３’（配列番号１３）
　シーケンシング用プライマー：
　５’−ＣＧＴＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧ−３’（配列番号１４）
　ＰＣＲ（反応スケール：２５μｌ）は鋳型として最終濃度０．６ｎＭのＤＮＡ断片を用いて実施し、９４℃−３０秒、４５℃−３０秒、６５℃−４分を１サイクルとして１５サイクル行った。反応液の最終組成は２０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．８）、１０ｍＭ　ＫＣｌ、１０ｍＭ（ＮＨ４）２ＳＯ４、２ｍＭ　ＭｇＳＯ４、０．１％　ＴｒｉｔｏｎＸ−１００、ＤｅｅｐＶｅｎｔ　ＤＮＡポリメラーゼ（０．０２ユニット／μｌ、ＮＥＢ）、１μＭ　５’側プライマー、１μＭ　３’側プライマー、０．３ｍＭ　各天然型塩基基質ｄＮＴＰ、２−１０μＭ　ｄＤｓｓＴＰ、および２−５０μＭ　ＮＨ２−ｈｘ−ｄＰｘＴＰである。１５サイクル後のＰＣＲ産物全長を変性ゲルで精製した後、ＤＮＡシーケンスの鋳型とし、シーケンシング解析をおこなった。 The template DNA and primer sequences used are as follows.
DNA S2 (55-mer, underlined primer annealing site)
5′- TTTCACACAGGAAAACAGCTATGAC GGCCCDsTTGCC CTATAGTGAGTCGTATTATC- 3 ′ (SEQ ID NO: 10)
Primer for PCR 5′-side primer: 5′-CGTTGTTAAAACGACGCCCAGGATAATACGACTCACTATAG-3 ′ (SEQ ID NO: 12)
3'-side primer: 5'-TTTCACACAGGAAACAGCCTATGAC-3 '(SEQ ID NO: 13)
Sequencing primers:
5′-CGTTGTTAAAACGACGCCAG-3 ′ (SEQ ID NO: 14)
PCR (reaction scale: 25 μl) was performed using a DNA fragment with a final concentration of 0.6 nM as a template, and 15 cycles were performed with 94 ° C. for 30 seconds, 45 ° C. for 30 seconds, and 65 ° C. for 4 minutes as one cycle. The final composition of the reaction solution was 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, Deep Vent DNA polymerase (0.02 unit / μl). NEB), 1 μM 5 ′ primer, 1 μM 3 ′ primer, 0.3 mM each natural base substrate dNTP, 2-10 μM dDssTP, and 2-50 μM NH 2 -hx-dPxTP. The full length of the PCR product after 15 cycles was purified with a denaturing gel, and then used as a template for a DNA sequence and subjected to sequencing analysis.
　ＤＮＡのシーケンシング反応は、全量２０μｌスケールにて、市販のＢｉｇＤｙｅ　Ｔｅｒｍｉｎａｔｏｒ　ｖ１．１　Ｃｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｋｉｔ　（Ａｐｐｌｉｅｄ　ＢｉｏＳｙｓｔｅｍｓ）のＣｙｃｌｅ　Ｓｅｑｕｅｎｃｉｎｇ　Ｍｉｘ　８μｌとプライマー（４ｐｍｏｌ）とＰＣＲ増幅されたＤＮＡ断片（およそ０．３ｐｍｏｌ程度）を混合し、４０ｐｍｏｌのｄＰａ’ＴＰもしくは１ｎｍｏｌのｄｄＰａ’ＴＰの存在下、２５サイクルのＰＣＲ（９６℃−１０秒、５０℃−５秒、６０℃−４分）を行った。未反応のダイダーミネーターをＣｅｎｔｒｉ−Ｓｅｐスピンカラム（Ａｐｐｌｉｅｄ　ＢｉｏＳｙｓｔｅｍｓ）で反応溶液からとり除き、残りの溶液を減圧吸引により乾燥した。残存物にＢｌｕｅ−Ｄｅｘｔｒａｎを希釈したホルムアミド溶液を４μｌ加えて懸濁し、その一部をＡＢＩ３７７ＤＮＡシーケンサーにより解析した。解析に用いたゲル組成は、７％ポリアクリルアミド—６Ｍ　尿素ゲルであり、配列のピークパターンは、Ａｐｐｌｉｅｄ　ＢｉｏＳｙｓｔｅｍｓ　ＰＲＩＳＭシークエンシング解析ｖ３．２ソフトウエアを用いて解析した。 The DNA sequencing reaction was performed on a commercially available BigDye Terminator v1.1 Cycle Sequencing Kit (Applied BioSystems) with a total volume of 20 μl, and a PCR amplification fragment (approx. 4 pmol) and about 0.3 pmol of PCR amplified fragment (about 4 pmol). ) And 25 cycles of PCR (96 ° C. for 10 seconds, 50 ° C. for 5 seconds, 60 ° C. for 4 minutes) in the presence of 40 pmol of dPa′TP or 1 nmol of ddPa′TP. Unreacted dye terminator was removed from the reaction solution with a Centri-Sep spin column (Applied BioSystems), and the remaining solution was dried by vacuum suction. The residue was suspended by adding 4 μl of a formamide solution diluted with Blue-Dextran, and a part thereof was analyzed by an ABI377 DNA sequencer. The gel composition used for the analysis was a 7% polyacrylamide-6M urea gel, and the peak pattern of the sequence was analyzed using Applied BioSystems PRISM sequencing analysis v3.2 software.
　実施例１０　Ｄｓｓ−Ｐｘ塩基対を用いたリアルタイムＰＣＲ（図１５）
　人工塩基Ｄｓｓを含むプライマーを用いて、ｄＰｘＴＰの基質存在下でＰＣＲを行った場合のリアルタイムＰＣＲの原理を図１４に示した。 Example 10 Real-time PCR using Dss-Px base pairs (FIG. 15)
FIG. 14 shows the principle of real-time PCR when PCR is performed in the presence of a dPxTP substrate using a primer containing the artificial base Dss.
　Ｄｓｓに対してＰｘが相補鎖に取り込まれると、ＰｘがＤｓｓのクエンチャー（消光剤）として作用するため、ＰＣＲ増幅された二本鎖ＤＮＡをＤｓｓの蛍光強度の減少から検出することができる。図１５は、実際に下記のＤＮＡ断片を用いて、リアルタイムＰＣＲを実施した結果であり、定量的な増幅プロットが得られ、反応液中（２５μｌ）に３コピーのＤＮＡでも検出可能であることがわかった。 When Px is incorporated into a complementary strand with respect to Dss, Px acts as a quencher (quenching agent) for Dss, and thus PCR-amplified double-stranded DNA can be detected from a decrease in fluorescence intensity of Dss. FIG. 15 shows the results of real-time PCR using the following DNA fragments. A quantitative amplification plot was obtained, and it was possible to detect even 3 copies of DNA in the reaction solution (25 μl). all right.
　実験に使用した配列（下線部はプライマーアニーリング部位に相当）
　５’−プライマー配列：５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣ−３’（配列番号１５）
　３’−プライマー配列：５’−ＡＡＴＡＡＴＧＣＤｓｓＴＣＣＴＣＡＡＡＧＧＴＧＧＴＧＡＣＴＴＣ−３’（配列番号１６）
　二本鎖鋳型ＤＮＡ（９８ｂｐ；片方の鎖のみ記載）：
　５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣＴＧＡＧＣＣＴＣＣＴＡＡＡＴＧＡＣＡＴＧＣＧＴＧＣＴＣＴＧＧＡＧＡＡＣＧＡＡＡＡＧＡＡＣＧＡＡＧＡＣＧＴＡＧＡＡＧＴＣＡＣＣＡＣＣＴＴＴＧＡＧＧＡ−３’（配列番号１７）
　具体的には、リアルタイムＰＣＲ装置（ストラタジーン、Ｍｘ３００５Ｐ）を用いて、プライマー各１μＭ、天然型塩基基質ｄＮＴＰ　各０．２ｍＭ、人工塩基基質ｄＰｘＴＰ　２μＭの存在下、９４℃−２分の後に、９４℃−５秒、６８℃−４０秒を１サイクルとする２ステップＰＣＲの条件で５５サイクルのＰＣＲを行った。ＰＣＲの反応スケールは２５μｌで、反応液の組成は４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ（ｐＨ８．０）、１６ｍＭ　ＫＣｌ、３．５ｍＭ　ＭｇＳＯ４、３．７５μｇ／ｍｌ　ＢＳＡ、１×ＴｉｔａｎｉｕｍＴａｑ　ＤＮＡポリメラーゼである。鋳型に用いたＤＮＡ断片は、反応液中にそれぞれ、０、３、１５、３０、１５０、３００、１５００、３０００、１５０００、３００００コピーになるように希釈し、それぞれの濃度につき、ＰＣＲを実施した。 Sequence used for experiment (underlined part corresponds to primer annealing site)
5′-primer sequence: 5′- CATGTAGATGCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15)
3′-primer sequence: 5′-AATAATGCDss TCCTCAAAGGGTGGACTTC- 3 ′ (SEQ ID NO: 16)
Double-stranded template DNA (98 bp; only one strand is listed):
5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17)
Specifically, using a real-time PCR apparatus (Stratagene, Mx3005P), in the presence of each primer 1 μM, natural base substrate dNTP 0.2 mM, artificial base substrate dPxTP 2 μM, after 94 ° C. for 2 minutes, 55 cycles of PCR were carried out under the conditions of 2-step PCR with 1 cycle of ℃ -5 seconds and 68 ° C-40 seconds. The reaction scale of PCR is 25 μl, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 μg / ml BSA, 1 × Titanium Taq DNA polymerase. The DNA fragment used for the template was diluted to 0, 3, 15, 30, 150, 300, 1500, 3000, 15000, 30000 copies in the reaction solution, and PCR was performed for each concentration. .
　検出に用いたフィルターセットは、励起３５０ｎｍ−蛍光４４０ｎｍ（ＡＬＥＸＡ用）である。データ解析には、Ｐｌｅｘｏｒ（登録商標）　ＡｎａｌｙＳｉＳ　Ｓｏｆｔｗａｒｅ　（ｖ１．５．４．１８，Ｐｒｏｍｅｇａ　＆　Ｅｒａｇｅｎ　ＢｉｏＳｃｉｅｎｃｅｓ）を用いた。結果を図１５に示した。 The filter set used for detection is excitation 350 nm-fluorescence 440 nm (for ALEXA). For data analysis, Plexor (registered trademark) AnalySiS Software (v1.5.4.18, Promega & Eragen BioSciences) was used. The results are shown in FIG.
　実施例１１　Ｄｓｓ−Ｐｎ塩基対を含むＤＮＡヘアピンの蛍光特性（図１６）
　Ｄｓｓを含む２種類のＤＮＡ、ｈａｉｒｐｉｎ　ｓｓＤＮＡ（３４−ｍｅｒ）（配列番号１８）とｓｓＤＮＡ（１２−ｍｅｒ）（配列番号１９）を１×ＥｘＴａｑ　Ｂｕｆｆｅｒ（ＴａＫａＲａ、２ｍＭ　ＭｇＣｌ２含有）中で１μＭとなるように調製し、Ｒｅｆｅｒｅｎｃｅ　ＤｙｅあるＲＯＸ（Ｉｎｖｉｔｒｏｇｅｎ）存在下（最終濃度１０００倍希釈）、温度変化による蛍光強度の変化をＭｘ３００５Ｐのｄｉｓｓｏｃｉａｔｉｏｎモードで検出した。ＲＯＸのシグナル強度で補正したのち、３５℃での値を基準として規格化したときのグラフを図１６に示した。 Example 11 Fluorescence properties of DNA hairpins containing Dss-Pn base pairs (FIG. 16)
2 types of DNA containing Dss, hairpin ssDNA (34-mer) (SEQ ID NO: 18) and ssDNA (12-mer) (SEQ ID NO: 19) become 1 μM in 1 × ExTaq Buffer (TaKaRa, containing 2 mM MgCl 2 ) In the presence of ROX (Invitrogen) as Reference Dye (final concentration diluted 1000 times), a change in fluorescence intensity due to a change in temperature was detected in a dissociation mode of Mx3005P. FIG. 16 shows a graph when the signal is normalized with the value at 35 ° C. after correction with the signal intensity of ROX.
　構造をもたない直鎖上のｓｓＤＮＡ（１２−ｍｅｒ）は、ＤＮＡを含まないＢｕｆｆｅｒのみ（バックグラウンド）と同様、少しずつ蛍光が現象するプロファイルであったのに対し、Ｄｓｓ−Ｐｎ塩基対を含むヘアピン構造を形成するｈａｉｒｐｉｎ　ｓｓＤＮＡ（３４−ｍｅｒ）は、温度上昇とともに蛍光が上昇するプロファイルが得られた。これは、低温状態ではヘアピン構造をとっているために消光作用のあるＰｎがＤｓｓと塩基対を形成し、Ｄｓｓの蛍光がＰｎで消光して蛍光強度が低くなっている一方で、温度を上げた場合にはヘアピン構造が壊れて消光作用がなくなり、Ｄｓｓの蛍光が検出されるようになったため、と考えられる。 The straight-line ssDNA (12-mer) having no structure was a profile in which fluorescence gradually occurred as in the case of only Buffer without DNA (background), whereas Dss-Pn base pair The hairpin ssDNA (34-mer), which forms the hairpin structure that was included, had a profile in which the fluorescence increased with increasing temperature. This is because Pn having a quenching action forms a base pair with Dss because it has a hairpin structure at low temperature, and the fluorescence intensity of Dss is quenched by Pn and the fluorescence intensity is lowered. In this case, the hairpin structure is broken, the quenching action is lost, and the fluorescence of Dss is detected.
　実施例１２　Ｄｓｓ−Ｐｎ塩基対を用いたモレキュラービーコンの可視化（図１７）
　各種ＤＮＡ断片、モレキュラービーコン（ＭＢ−Ｃ、２６−ｍｅｒ）（配列番号２０）とＴａｒｇｅｔ　ＤＮＡ（７１Ｇ、７１−ｍｅｒ）（配列番号２１）をそれぞれ２μＭとなるように調製し、等量（５０μｌずつ）混合した。ネガティブコントロールとして、Ｔａｒｇｅｔ　ＤＮＡを含まない溶液を、ＭＢ−Ｃ溶液と混合した。最終的な溶液組成はＤＮＡ濃度各１μＭ、１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０）、１００ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＴＡである。この溶液をＰＣＲマシンにて９０℃で１０秒間加熱後、２５℃まで徐冷した。励起波長３７５ｎｍのＵＶ−ＬＥＤランプによる照射下もしくは自然光下にて、デジタルカメラにより撮影した写真を図１７の右に示した。 Example 12 Visualization of molecular beacons using Dss-Pn base pairs (FIG. 17)
Various DNA fragments, molecular beacon (MB-C, 26-mer) (SEQ ID NO: 20) and Target DNA (71G, 71-mer) (SEQ ID NO: 21) were prepared to 2 μM each, and equal amounts (50 μl each) ) Mixed. As a negative control, a solution containing no Target DNA was mixed with an MB-C solution. The final solution composition is DNA concentration 1 μM each, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA. This solution was heated at 90 ° C. for 10 seconds with a PCR machine and then gradually cooled to 25 ° C. A photograph taken with a digital camera under irradiation with a UV-LED lamp with an excitation wavelength of 375 nm or under natural light is shown on the right side of FIG.
　ターゲットＤＮＡ非存在下では、モレキュラービーコンはループ—ステム構造を形成して、Ｄｓｓ−Ｐｎ塩基対形成により、Ｄｓｓの蛍光が消光されるが、ターゲットＤＮＡ存在下では、モレキュラービーコンのループ部分がハイブリダイゼーションによりターゲットＤＮＡと二本鎖を形成し、ステム構造が壊れてＤｓｓ−Ｐｎ塩基対が形成なくなり、Ｄｓｓの蛍光が検出されることが目視にて確認できた。 In the absence of the target DNA, the molecular beacon forms a loop-stem structure, and the Dss fluorescence is quenched by Dss-Pn base pairing. However, in the presence of the target DNA, the molecular beacon loop part is hybridized. As a result, a double strand was formed with the target DNA, the stem structure was broken, and the Dss-Pn base pair was not formed, and it was confirmed visually that Dss fluorescence was detected.
　実施例１３　Ｄｓｓ−Ｐｎ塩基対を用いたモレキュラービーコンによる一塩基変異の検出（図１８）
　５００ｎＭに希釈したモレキュラービーコン（２６−ｍｅｒ、ＭＢ−Ｃ（配列番号２０）またはＭＢ−Ｔ（配列番号２３））の溶液を５０μｌずつ分注し、最終溶液の５倍濃度のＴａｒｇｅｔ　ＤＮＡ断片（７１−ｍｅｒ、７１Ｇ（配列番号２１）または７１Ａ（配列番号２２）、１２．５μｌ）と混合したサンプルをインキュベーターにて４５℃で５分以上保温して平衡状態とした。蛍光測定はＪＡＳＣＯ　ＦＰ−６５００　スペクトロメーターを用いて行い、セルに溶液を移したのち装置中（４５℃設定）で２分放置後に、自動シャッター制御を使用して３９０ｎｍで励起し４３０−４７０ｎｍの蛍光スペクトルを測定した。最終溶液中の組成は、モレキュラービーコン　４００ｎＭ、Ｔａｒｇｅｔ　ＤＮＡ　０−３２００ｎＭ、１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７．０）、１００ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＴＡである。 Example 13 Detection of single nucleotide mutation by molecular beacon using Dss-Pn base pair (FIG. 18)
A 50 μl aliquot of a molecular beacon (26-mer, MB-C (SEQ ID NO: 20) or MB-T (SEQ ID NO: 23)) diluted to 500 nM was dispensed, and the Target DNA fragment (71 A sample mixed with -mer, 71G (SEQ ID NO: 21) or 71A (SEQ ID NO: 22), 12.5 μl) was kept in an incubator at 45 ° C. for 5 minutes or more to obtain an equilibrium state. Fluorescence measurement was performed using a JASCO FP-6500 spectrometer. After transferring the solution to the cell, it was left in the apparatus (at 45 ° C.) for 2 minutes, then excited at 390 nm using automatic shutter control, and 430-470 nm fluorescence. The spectrum was measured. The composition in the final solution is molecular beacon 400 nM, Target DNA 0-3200 nM, 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM EDTA.
　図１８に示したグラフは、４５４ｎｍの蛍光強度について、Ｔａｒｇｅｔ　ＤＮＡ断片非存在下での蛍光強度でそれぞれ規格化してからプロットしたものである。一塩基のミスマッチがある場合には完全にマッチした場合よりも蛍光強度が有意に低くなることを利用して、一塩基変異をＤｓｓ−Ｐｎ塩基対を利用したモレキュラービーコンにより検出できることがわかった。 The graph shown in FIG. 18 is plotted after standardizing the fluorescence intensity at 454 nm with the fluorescence intensity in the absence of the Target DNA fragment. It was found that a single base mutation can be detected by a molecular beacon using a Dss-Pn base pair by utilizing the fact that the fluorescence intensity is significantly lower when there is a single base mismatch than when it is a perfect match.
　実施例１４　Ｃｙ３−Ｐｘ／Ｄｓｓ塩基対を用いた可視化ＰＣＲ（図２０）
　人工塩基Ｄｓｓを含むプライマーを用いて、Ｃｙ３−ｈｘ−ｄＰｘＴＰの基質存在下でＰＣＲを行った場合のリアルタイムＰＣＲの原理を図１９に示した。Ｄｓｓに対してＣｙ３−ｈｘ−ｄＰｘが相補鎖に取り込まれると、３５０ｎｍ付近の照射でＤｓｓとＣｙ３間にＦＲＥＴが起こるため、ＰＣＲ増幅された二本鎖ＤＮＡを特異的に光らせることができる。また、このＦＲＥＴによる蛍光は目視検出が可能なこともわかった（図２０）。 Example 14 Visualization PCR Using Cy3-Px / Dss Base Pair (FIG. 20)
FIG. 19 shows the principle of real-time PCR when PCR is performed in the presence of a substrate of Cy3-hx-dPxTP using a primer containing the artificial base Dss. When Cy3-hx-dPx is incorporated into a complementary strand with respect to Dss, FRET occurs between Dss and Cy3 by irradiation at around 350 nm, and thus PCR-amplified double-stranded DNA can be specifically illuminated. It was also found that the fluorescence by FRET can be detected visually (FIG. 20).
　実験に用いた配列は図１５と同一である。
　実験に使用した配列　（下線部はプライマーアニーリング部位に相当）
　５’−プライマー配列　：５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣ−３’（配列番号１５）
　３’−プライマー配列　：５’−ＡＡＴＡＡＴＧＣＤｓｓＴＣＣＴＣＡＡＡＧＧＴＧＧＴＧＡＣＴＴＣ−３’（配列番号１６）
　二本鎖鋳型ＤＮＡ　（９８　ｂｐ；片方の鎖のみ記載）：
　５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣＴＧＡＧＣＣＴＣＣＴＡＡＡＴＧＡＣＡＴＧＣＧＴＧＣＴＣＴＧＧＡＧＡＡＣＧＡＡＡＡＧＡＡＣＧＡＡＧＡＣＧＴＡＧＡＡＧＴＣＡＣＣＡＣＣＴＴＴＧＡＧＧＡ−３’（配列番号１７）
　具体的には、リアルタイムＰＣＲ装置（ストラタジーン、Ｍｘ３００５Ｐ）を用いて、プライマー各１μＭ、天然型塩基基質ｄＮＴＰ各０．２ｍＭ、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ　２μＭの存在下、９４℃−２分の後、９４℃−５秒、６８℃−４０秒を１サイクルとする２ステップＰＣＲの条件で５５サイクルのＰＣＲを行った。ＰＣＲの反応スケールは２５μｌで、反応液の組成は４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ（ｐＨ８．０）、１６ｍＭ　ＫＣｌ、３．５ｍＭ　ＭｇＳＯ４、３．７５μｇ／ｍｌ　ＢＳＡ、１×ＴｉｔａｎｉｕｍＴａｑ　ＤＮＡポリメラーゼである。鋳型に用いたＤＮＡ断片は、反応液中にそれぞれ、０、３、３０、３００、３０００、３００００、３０００００、３００００００コピーになるように希釈し、それぞれの濃度につき、ＰＣＲを実施した。その反応チューブを直接、３６５ｎｍのＵＶで照射し、オレンジフィルターを通して目視で蛍光を検出した。 The arrangement | sequence used for experiment is the same as FIG.
Sequence used in the experiment (underlined part corresponds to the primer annealing site)
5′-primer sequence: 5′- CATGTAGATGCCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15)
3′-primer sequence: 5′-AATAATGCDss TCCTCAAAGGTGGTACTACT- 3 ′ (SEQ ID NO: 16)
Double-stranded template DNA (98 bp; only one strand is listed):
5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17)
Specifically, using a real-time PCR apparatus (Stratagene, Mx3005P), 94 ° C.-2 minutes in the presence of each primer 1 μM, natural base substrate dNTP 0.2 mM, artificial base substrate Cy3-hx-dPxTP 2 μM Thereafter, 55 cycles of PCR were performed under the conditions of 2-step PCR with 94 ° C. for 5 seconds and 68 ° C. for 40 seconds as one cycle. The reaction scale of PCR is 25 μl, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 μg / ml BSA, 1 × Titanium Taq DNA polymerase. The DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration. The reaction tube was directly irradiated with 365 nm UV, and fluorescence was detected visually through an orange filter.
　実施例１５　蛍光性分子Ｃｙ３を結合した消光性Ｐｘ塩基によるリアルタイムＰＣＲ（図２２）
　人工塩基Ｄｓを含むプライマーを用いて、蛍光性分子（Ｃｙ３など）を結合したｄＰｘＴＰ誘導体の基質存在下でＰＣＲを行った場合のリアルタイムＰＣＲの原理を図２１に示した。蛍光性分子を、消光性のＰｘ塩基に結合させると、蛍光性分子の蛍光が３０％程度消光する。これを基質（Ｃｙ３−ｈｘ−ｄＰｘＴＰ）　にして、Ｄｓ塩基を導入したプライマーを用いたＰＣＲを行うと、Ｃｙ３−ｈｘ−ＰｘがＤＮＡ中に取り込まれることで、Ｃｙ３の蛍光強度が増大する。図２２は、実際に下記のＤＮＡ断片を用いて、リアルタイムＰＣＲを実施した結果であり、定量的な増幅プロットが得られ、反応液中（２５μｌ）に３コピーのＤＮＡでも検出可能であることがわかった。 Example 15 Real-time PCR with quenching Px base bound to the fluorescent molecule Cy3 (FIG. 22)
FIG. 21 shows the principle of real-time PCR when PCR is performed in the presence of a substrate of a dPxTP derivative to which a fluorescent molecule (such as Cy3) is bound using a primer containing an artificial base Ds. When a fluorescent molecule is bound to a quenching Px base, the fluorescence of the fluorescent molecule is quenched by about 30%. When this is used as a substrate (Cy3-hx-dPxTP) and PCR is carried out using a primer into which a Ds base has been introduced, Cy3-hx-Px is incorporated into DNA, thereby increasing the fluorescence intensity of Cy3. FIG. 22 shows the results of real-time PCR using the following DNA fragments. A quantitative amplification plot was obtained, and it was possible to detect even 3 copies of DNA in the reaction solution (25 μl). all right.
　実験に使用した配列（下線部はプライマーアニーリング部位に相当）
　５’−プライマー配列：５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣ−３’（配列番号１５）
　３’−プライマー配列：５’−ＡＡＴＡＡＴＧＣＤｓＴＣＣＴＣＡＡＡＧＧＴＧＧＴＧＡＣＴＴＣ−３’（配列番号２４）
　二本鎖鋳型ＤＮＡ（９８ｂｐ；片方の鎖のみ記載）：
　５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣＴＧＡＧＣＣＴＣＣＴＡＡＡＴＧＡＣＡＴＧＣＧＴＧＣＴＣＴＧＧＡＧＡＡＣＧＡＡＡＡＧＡＡＣＧＡＡＧＡＣＧＴＡＧＡＡＧＴＣＡＣＣＡＣＣＴＴＴＧＡＧＧＡ−３’（配列番号１７）
　具体的には、リアルタイムＰＣＲ装置　（ストラタジーン、Ｍｘ３００５Ｐ）を用いて、プライマー各１μＭ、天然型塩基基質ｄＮＴＰ各０．２ｍＭ、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ　２μＭの存在下、９４℃−２分の後、９４℃−５秒、６８℃−４０秒を１サイクルとする２ステップＰＣＲの条件で５５サイクルのＰＣＲを行った。ＰＣＲの反応スケールは２５μｌで、反応液の組成は４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ（ｐＨ８．０）、１６ｍＭ　ＫＣｌ、３．５ｍＭ　ＭｇＳＯ４、３．７５μｇ／ｍｌ　ＢＳＡ、１×ＴｉｔａｎｉｕｍＴａｑＤＮＡポリメラーゼである。鋳型に用いたＤＮＡ断片は、反応液中にそれぞれ、０、３、３０、３００、３０００、３００００、３０００００、３００００００コピーになるように希釈し、それぞれの濃度につき、ＰＣＲを実施した。検出に用いたフィルターセットは、励起５４５ｎｍ−蛍光５６８ｎｍ（ＣＹ３用）である。データ解析には、付属の解析ソフトＭｘＰｒｏ　ｖｅｒｓｉｏｎ　４．１０を用いた。 Sequence used for experiment (underlined part corresponds to primer annealing site)
5′-primer sequence: 5′- CATGTAGATGCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15)
3′-primer sequence: 5′-AATAATGCDs TCCTCAAAGGTGGTACTACT- 3 ′ (SEQ ID NO: 24)
Double-stranded template DNA (98 bp; only one strand is listed):
5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17)
Specifically, using a real-time PCR apparatus (Stratagene, Mx3005P), 94 ° C.-2 minutes in the presence of each primer 1 μM, natural base substrate dNTP 0.2 mM, artificial base substrate Cy3-hx-dPxTP 2 μM After that, 55 cycles of PCR were performed under the conditions of 2-step PCR with 94 ° C. for 5 seconds and 68 ° C. for 40 seconds as one cycle. The PCR reaction scale is 25 μl, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 μg / ml BSA, 1 × Titanium Taq DNA polymerase. The DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration. The filter set used for detection is excitation 545 nm-fluorescence 568 nm (for CY3). For data analysis, attached analysis software MxPro version 4.10 was used.
　実施例１６　蛍光性分子Ｃｙ３を結合した消光性Ｐｘ塩基によるリアルタイムＰＣＲ産物のゲル電気泳動上での検出（図２３）
　図２２に示したＰＣＲ産物は、Ｃｙ３が取り込まれているため、この産物をアガロースゲルで電気泳動した場合、従来のＥｔＢｒやＳＹＢＲ　ＧｒｅｅｎなどのＤＮＡ染色色素を用いることなく、ゲル上でＰＣＲ産物をＣｙ３の蛍光で検出可能である。図２３では、図２２に示したＰＣＲ産物１２μｌを、４％アガロースゲルで電気泳動し、バイオイメージングアナライザー、ＦＬＡ７０００（富士フィルム）のＣｙ３検出モード（励起レーザー：５３２ｎｍ、検出フィルター：Ｏ５８０）でバンドパターンを検出した結果を示した。 Example 16 Detection of real-time PCR product on gel electrophoresis with quenching Px base bound to fluorescent molecule Cy3 (FIG. 23)
Since the PCR product shown in FIG. 22 incorporates Cy3, when this product is electrophoresed on an agarose gel, the PCR product can be run on the gel without using a conventional DNA staining dye such as EtBr or SYBR Green. Detection is possible with Cy3 fluorescence. In FIG. 23, 12 μl of the PCR product shown in FIG. 22 was electrophoresed on a 4% agarose gel, and a band pattern was obtained using Cy3 detection mode (excitation laser: 532 nm, detection filter: O580) of a bioimaging analyzer, FLA7000 (Fuji Film). The result of detecting was shown.
　実施例１７　蛍光性分子　Ｃｙ３　ならびに蛍光性人工塩基Ｓを含むＤＮＡの蛍光特性（図２４）
　化学合成後ＨＰＬＣ精製したＤＮＡ断片を最終濃度が５μＭとなるように、１００ｍＭ　ＮａＣｌ、０．１ｍＭ　ＥＤＴＡを含む１０ｍＭ　リン酸ナトリウム緩衝液（ｐＨ７）で調製し、これらの溶液の蛍光特性を目視ならびに蛍光スペクトルで調べた結果を図２４に示した。 Example 17 Fluorescence characteristics of DNA containing fluorescent molecule Cy3 and fluorescent artificial base S (FIG. 24)
DNA fragments purified by HPLC after chemical synthesis were prepared with 10 mM sodium phosphate buffer (pH 7) containing 100 mM NaCl and 0.1 mM EDTA so that the final concentration would be 5 μM. The results of examining the spectrum are shown in FIG.
　ＵＶ照射は、ＵＶトランスイルミネーターを用いて、下方より行った。蛍光性人工塩基ｓを一つ含むＤＮＡ断片は、２５４ｎｍ、３０２ｎｍ、３６５ｎｍの照射で発光し（写真レーン２）、ｓを２つ隣接してＤＮＡ中に導入すると、その蛍光が消光した（写真レーン３）。Ｃｙ３を導入したＤＮＡは、２５４ｎｍ、３０２ｎｍの照射で少し発光するが、３６５ｎｍの照射ではほとんど発光しなかった（写真レーン４）。これに対し、ＤＮＡ中のＣｙ３の近傍に、ｓを１つあるいは２つ導入するとＣｙ３の蛍光が観測され、ＦＲＥＴが起こることが確認できた（写真レーン５−７）。溶液を３６５ｎｍで励起した場合の蛍光スペクトルをグラフに示した。 UV irradiation was performed from below using a UV transilluminator. A DNA fragment containing one fluorescent artificial base s emits light when irradiated at 254 nm, 302 nm, and 365 nm (photo lane 2), and when two s are introduced into the DNA adjacent to each other, the fluorescence is quenched (photo lane). 3). The DNA into which Cy3 was introduced emitted little light when irradiated with 254 nm and 302 nm, but hardly emitted when irradiated with 365 nm (Photo Lane 4). On the other hand, when one or two s were introduced in the vicinity of Cy3 in the DNA, Cy3 fluorescence was observed, confirming that FRET occurred (photo lanes 5-7). The graph shows the fluorescence spectrum when the solution was excited at 365 nm.
　実施例１８　蛍光性分子　Ｃｙ３を結合した消光性のＰｘ塩基と蛍光性人工塩基Ｓを組み合わせた可視化ＰＣＲ法（図２６−２８）
　人工塩基Ｄｓおよび蛍光性人工塩基ｓを隣接して２つ含むプライマーを用いて、Ｃｙ３−ｈｘ−ｄＰｘＴＰ基質存在下でＰＣＲを行った場合のリアルタイムＰＣＲの原理を図２５に示した。ｓを隣接して２つ導入するとｓの蛍光は完全に消光する。しかし、Ｓの近傍にＤｓを配置し、そのＤｓにＣｙ３−ｈｘが相補して特異的に組み込まれると、３６５ｎｍ付近の照射でＳとＣｙ３間にＦＲＥＴが起こり、ＰＣＲ増幅された二本鎖ＤＮＡのみを特異的に光らせることができる。 Example 18 Visualization PCR Method Combining Quenching Px Base with Fluorescent Molecule Cy3 and Fluorescent Artificial Base S (FIGS. 26-28)
FIG. 25 shows the principle of real-time PCR when PCR is performed in the presence of a Cy3-hx-dPxTP substrate using a primer containing two adjacent artificial bases Ds and fluorescent artificial bases s. When two s are introduced adjacent to each other, the fluorescence of s is completely quenched. However, when Ds is arranged in the vicinity of S and Cy3-hx is complemented and specifically incorporated in the Ds, FRET occurs between S and Cy3 by irradiation around 365 nm, and PCR amplified double-stranded DNA Only can be shined specifically.
　図２６は、実際に下記のＤＮＡ断片を用いて、ＰＣＲ２５サイクルを行った産物を目視で確認した結果である。図２６のｓｓ−Ｃｙ３を利用した系では、ＰＣＲ装置　（ＭＪリサーチ、ＰＴＣ−１００）を用いて、プライマー各１μＭ、天然型塩基基質ｄＮＴＰ各０．２　ｍＭ、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ　２μＭの存在下、９４℃−２分の後、９４℃−５秒、６８℃−４０秒を１サイクルとする２ステップＰＣＲの条件で２５サイクルのＰＣＲを行った。ＰＣＲの反応スケールは２５μｌで、反応液の組成は４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ　（ｐＨ　８．０）、１６ｍＭ　ＫＣｌ、３．５ｍＭ　ＭｇＳＯ４、３．７５μｇ／ｍｌ　ＢＳＡ、１×ＴｉｔａｎｉｕｍＴａｑ　ＤＮＡポリメラーゼである。鋳型に用いたＤＮＡ断片濃度は０．５　ｎＭである。従来法であるＳＹＢＲ　Ｇｒｅｅｎ　Ｉ　存在下でのＰＣＲでは、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ　２μＭの代わりに、ＳＹＢＲ　Ｇｒｅｅｎ　Ｉ（最終濃度１／３００００）、レファレンス色素としてＲＯＸ（最終濃度　１／５００）を加えている。 FIG. 26 shows the result of visually confirming the product that was subjected to PCR 25 cycles using the following DNA fragment. In the system using ss-Cy3 in FIG. 26, using a PCR apparatus (MJ Research, PTC-100), each primer 1 μM, natural base substrate dNTP each 0.2 mM, artificial base substrate Cy3-hx-dPxTP 2 μM In the presence of the sample, 25 cycles of PCR were performed under the conditions of 2-step PCR, with 94 ° C for 5 minutes and 68 ° C for 40 seconds after 94 ° C for 2 minutes. The reaction scale of PCR is 25 μl, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 μg / ml BSA, 1 × Titanium Taq DNA polymerase. The DNA fragment concentration used for the template is 0.5 nM. In the conventional PCR in the presence of SYBR Green I, instead of the artificial base substrate Cy3-hx-dPxTP 2 μM, SYBR Green I (final concentration 1/30000) and ROX (final concentration 1/500) as a reference dye are used. Added.
　ＳＹＢＲ　Ｇｒｅｅｎ　Ｉ存在下でのリアルタイムＰＣＲ検出は最もよく用いられる方法の一つであるが、図２６中、右側２レーンの写真に示したとおり、ＤＮＡ有無での蛍光変化が大きくないために、目視での検出は難しい。図２６の左側２レーンに示した通り、本法ではＰＣＲ検出を目視での検出が可能である。 Real-time PCR detection in the presence of SYBR Green I is one of the most commonly used methods, but as shown in the photograph on the right two lanes in FIG. Detection with is difficult. As shown in the two lanes on the left side of FIG. 26, this method enables visual detection of PCR detection.
　図２７ａに示したリアルタイムＰＣＲでは、リアルタイムＰＣＲ装置（ストラタジーン、Ｍｘ３００５Ｐ）を用いて、プライマー　各１μＭ、天然型塩基基質ｄＮＴＰ　各０．２　ｍＭ、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ　２μＭの存在下、９４℃−２分の後、９４℃−５秒、６８℃−４０秒を１サイクルとする２ステップＰＣＲの条件で５５サイクルのＰＣＲを行った。ＰＣＲの反応スケールは２５μｌで、反応液の組成は４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ　ｐＨ８．０）、１６ｍＭ　ＫＣｌ、３．５ｍＭ　ＭｇＳＯ４、３．７５μｇ／ｍｌ　ＢＳＡ、１×ＴｉｔａｎｉｕｍＴａｑ　ＤＮＡポリメラーゼである。鋳型に用いたＤＮＡ断片は、反応液中にそれぞれ、０、３、３０、３００、３０００、３００００、３０００００、３００００００コピーになるように希釈し、それぞれの濃度につき、ＰＣＲを実施した。 In the real-time PCR shown in FIG. 27a, using a real-time PCR apparatus (Stratagene, Mx3005P), in the presence of each primer 1 μM, natural base substrate dNTP 0.2 mM, artificial base substrate Cy3-hx-dPxTP 2 μM, After 94 ° C. for 2 minutes, 55 cycles of PCR were performed under the conditions of 2-step PCR with 94 ° C. for 5 seconds and 68 ° C. for 40 seconds as one cycle. The PCR reaction scale is 25 μl, and the composition of the reaction solution is 40 mM Tricine-KOH pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 μg / ml BSA, 1 × Titanium Taq DNA polymerase. The DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration.
　さらに、図２７ａに示したように、反応液中（２５μｌ）に３コピーのＤＮＡでも３６５ｎｍ照射により、ＰＣＲ産物を目視で検出可能であることがわかった。
　また、図２８は図２７ａの可視化ＰＣＲ産物をアガロースゲルで電気泳動した場合で、３１２ｎｍ照射でｓからＣｙ３へのＦＲＥＴにより産物検出ができること、また５３２ｎｍ照射でＤＮＡ中に直接取り込まれたＣｙ３の蛍光により産物検出ができることが確認できた。 Furthermore, as shown in FIG. 27a, it was found that even with 3 copies of DNA in the reaction solution (25 μl), the PCR product could be detected visually by irradiation at 365 nm.
FIG. 28 shows the case where the visualized PCR product of FIG. 27a is electrophoresed on an agarose gel, and the product can be detected by FRET from s to Cy3 by 312 nm irradiation, and the fluorescence of Cy3 directly incorporated into DNA by 532 nm irradiation. It was confirmed that product detection was possible.
　実験に使用した配列（下線部はプライマーアニーリング部位に相当）
　５’−プライマー配列：５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣ−３’（配列番号１５）
　３’−プライマー配列：５’−ＡＡＴＡＡＳＳＧＣＤｓＴＣＣＴＣＡＡＡＧＧＴＧＧＴＧＡＣＴＴＣ−３’（配列番号２５）
　二本鎖鋳型ＤＮＡ　（９８　ｂｐ；片方の鎖のみ記載）：
　５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣＴＧＡＧＣＣＴＣＣＴＡＡＡＴＧＡＣＡＴＧＣＧＴＧＣＴＣＴＧＧＡＧＡＡＣＧＡＡＡＡＧＡＡＣＧＡＡＧＡＣ
　ＧＴＡＧＡＡＧＴＣＡＣＣＡＣＣＴＴＴＧＡＧＧＡ−３’（配列番号１７）
　図２８では、図２７ａに示したＰＣＲ産物１２μｌを、４％アガロースゲルで電気泳動し、バイオイメージングアナライザー、ＬＡＳ４０００　（富士フィルム）のＥｔＢｒ検出モード（励起：３１２ｎｍ透過ＵＶ、検出フィルター　６０５ＤＦ４０）を用いてｓとＣｙ３間のＦＲＥＴにより産物を検出した結果、およびＦＬＡ７０００　（富士フィルム）
　のＣｙ３検出モード（励起レーザー：５３２ｎｍ、検出フィルター：Ｏ５８０）でＣｙ３の蛍光から直接産物を検出した結果を示した。 Sequence used for experiment (underlined part corresponds to primer annealing site)
5′-primer sequence: 5′- CATGTAGATGCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15)
3'-primer sequence: 5'-AATAASSGCDs TCCTCAAAGGTGGTGACTTC- 3 '(SEQ ID NO: 25)
Double-stranded template DNA (98 bp; only one strand is listed):
5'- CATGTAGATGCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAGAGAACGAAGAC
GTA GAAGTCACCACCCTTTGAGA- 3 ′ (SEQ ID NO: 17)
In FIG. 28, 12 μl of the PCR product shown in FIG. 27a was electrophoresed on a 4% agarose gel, and the bioimaging analyzer, LAS4000 (Fuji Film) EtBr detection mode (excitation: 312 nm transmission UV, detection filter 605DF40) was used. Results of product detection by FRET between s and Cy3, and FLA7000 (Fuji Film)
The results of directly detecting the product from the fluorescence of Cy3 in the Cy3 detection mode (excitation laser: 532 nm, detection filter: O580) are shown.
　実施例１９　蛍光性分子　Ｃｙ３　を結合した消光性のＰｘ塩基と蛍光性人工塩基ｓを組み合わせた可視化ＰＣＲ法：　各ＰＣＲサイクルの蛍光強度の定量）（図２７ｂ−図２７ｄ）
　本実施例は、図２７ａの実験の追実施例である。 Example 19 Visualization PCR Method Combining Quenching Px Base Coupled with Fluorescent Molecule Cy3 and Fluorescent Artificial Base s: Quantification of Fluorescence Intensity in Each PCR Cycle) (FIGS. 27b-27d)
This example is a supplementary example of the experiment of FIG. 27a.
　人工塩基Ｄｓおよび蛍光性人工塩基ｓを隣接して２つ含むプライマーを用いて、Ｃｙ３−ｈｘ−ｄＰｘＴＰ基質存在下でＰＣＲを行った場合、増幅されたＤＮＡ中のＣｙ３の蛍光強度の増加を測定することにより、リアルタイムＰＣＲ（図２７ｂ）にも利用できた。また、異なる初濃度のＤＮＡのＰＣＲ増幅を目視での検出（図２７ｃ）も可能になった。さらには、ＰＣＲサイクル毎で、その増幅の過程をチューブの撮影画像を処理して、ＤＮＡの増幅を定量化することもできた（図２７ｄ）。 When PCR was performed in the presence of Cy3-hx-dPxTP substrate using a primer containing two adjacent artificial bases Ds and fluorescent artificial base s, the increase in fluorescence intensity of Cy3 in the amplified DNA was measured. By doing so, it could also be used for real-time PCR (FIG. 27b). In addition, it is possible to visually detect PCR amplification of different initial concentrations of DNA (FIG. 27c). Furthermore, the amplification process of the DNA could be quantified by processing the captured image of the tube during each PCR cycle (FIG. 27d).
　実験に使用した配列（下線部はプライマーアニーリング部位に相当）
　５’−プライマー配列　：５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣ−３’（配列番号１５）
　３’−プライマー配列：５’−　ＡＡＴＡＡｓｓＧＣＤｓＴＣＣＴＣＡＡＡＧＧＴＧＧＴＧＡＣＴＴＣ−３’（配列番号２６）
　二本鎖鋳型ＤＮＡ（９８ｂｐ；片方の鎖のみ記載）：
　５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣＴＧＡＧＣＣＴＣＣＴＡＡＡＴＧＡＣＡＴＧＣＧＴＧＣＴＣＴＧＧＡＧＡＡＣＧＡＡＡＡＧＡＡＣＧＡＡＧＡＣＧＴＡＧＡＡＧＴＣＡＣＣＡＣＣＴＴＴＧＡＧＧＡ−３’（配列番号１７）
　ＰＣＲ反応は、リアルタイムＰＣＲ装置（ストラタジーン、Ｍｘ３００５Ｐ）を用いて、プライマー　各１μＭ、天然型塩基基質ｄＮＴＰ　各０．２ｍＭ、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ　２μＭの存在下、９４℃−２分の後、９４℃−５秒、６８℃−４０秒を１サイクルとする２ステップＰＣＲの条件で３０、３５、４０、４５、５５サイクルのＰＣＲを行った。 Sequence used for experiment (underlined part corresponds to primer annealing site)
5′-primer sequence: 5′- CATGTAGATGCCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15)
3′-primer sequence: 5′-AATAAssGCDs TCCTCAAAGGTGGTGACTTC- 3 ′ (SEQ ID NO: 26)
Double-stranded template DNA (98 bp; only one strand is listed):
5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17)
The PCR reaction was carried out using a real-time PCR apparatus (Stratagene, Mx3005P) at 94 ° C. for 2 minutes in the presence of each primer 1 μM, natural base substrate dNTP 0.2 mM, and artificial base substrate Cy3-hx-dPxTP 2 μM. Thereafter, PCR was performed for 30, 35, 40, 45, and 55 cycles under conditions of 2-step PCR with 94 ° C. for 5 seconds and 68 ° C. for 40 seconds as one cycle.
　ＰＣＲの反応スケールは２５μｌで、反応液の組成は４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ（ｐＨ８．０）、１６ｍＭ　ＫＣｌ、３．５ｍＭ　ＭｇＳＯ４、３．７５μｇ／ｍｌ　ＢＳＡ、１×ＴｉｔａｎｉｕｍＴａｑ　ＤＮＡポリメラーゼである。鋳型に用いたＤＮＡ断片は、反応液中にそれぞれ、０、３、３０、３００、３０００、３００００、３０００００、３００００００コピーになるように希釈し、それぞれの濃度につき、ＰＣＲを実施した。 The reaction scale of PCR is 25 μl, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4 , 3.75 μg / ml BSA, 1 × Titanium Taq DNA polymerase. The DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration.
　反応終了後のチューブでの画像処理による定量解析は、以下の手順で行った。ＵＶトランスイルミネーターを用いて、下方より３６５ｎｍのＵＶ照射下で、ＵＶカットフィルターおよびオレンジフィルター越しにデジタルカメラにより撮影し、得られたファイル（ＪＰＥＧ形式）をＡｄｏｂｅ　Ｐｈｏｔｏｓｈｏｐ　ｖｅｒ．６．０で、画像モードをグレースケール、解像度を７２ｐｉｘｅｌ／ｉｎｃｈとしてＴｉｆｆ形式ファイルに変換した。このファイルを、Ｓｃｉｅｎｃｅ　Ｌａｂ　２００５　Ｍｕｌｔｉ　Ｇａｕｇｅソフトウエアで読み込み、定量解析を行った。具体的には、チューブの反応溶液部分［１０１５（ｐｉｘｅｌ）２］のＱｕａｎｔｕｍ　Ｌｅｖｅｌ（ＱＬ値）から、バックグラウンドの値（チューブ間のエリア、７カ所の平均値）を差し引き、その単位面積あたりの値を、それぞれＰＣＲサイクルに対して、あるいは鋳型に用いたコピー数に対して、プロットした結果をグラフに示した。 Quantitative analysis by image processing in the tube after completion of the reaction was performed according to the following procedure. Using a UV transilluminator, the image was taken with a digital camera through a UV cut filter and an orange filter under UV irradiation of 365 nm from below, and the resulting file (JPEG format) was taken from Adobe Photoshop ver. At 6.0, the image mode was converted to a Tiff format file with a gray scale and a resolution of 72 pixels / inch. This file was read with Science Lab 2005 Multi Gauge software for quantitative analysis. Specifically, from the Quantum Level (QL value) of the reaction solution portion [1015 (pixel) 2 ] of the tube, the background value (area between tubes, average value of 7 locations) is subtracted, The results are plotted on the graph for each PCR cycle or copy number used for the template.
　結果を図２７ｂ−ｄに示す。
　実施例２０　蛍光性分子（ｓ塩基）を天然型塩基にリンカーを介して結合したヌクレオシド誘導体（ｓ−ｈｘ−ｄＵ）とＤｓ−Ｐｘ塩基対を用いたＰＣＲ産物の検出法（図２９ｂ−図２９ｄ）
　本実施例は図２９ａの対実施例である。 The results are shown in FIGS. 27b-d.
Example 20 PCR product detection method using a nucleoside derivative (s-hx-dU) in which a fluorescent molecule (s base) is bound to a natural base via a linker and a Ds-Px base pair (FIGS. 29b to 29d) )
This example is a counter example of FIG. 29a.
　蛍光性人工塩基であるｓをリンカーを介して天然型塩基Ｕに結合した修飾塩基（ｓ−ｈｘ−ｄＵ）を隣接して２つ含むプライマーを用いて、Ｃｙ３−ｈｘ−ｄＰｘＴＰ基質存在下でＰＣＲを行った場合のリアルタイムＰＣＲの原理を図２９ａに示した。ｓ−ｈｘ−ｄＵを隣接して２つ導入すると、ｓを隣接して２つ導入した場合と同様に（図２５）、ｓの蛍光は消光される。しかし、ｓ−ｈｘ−ｄＵの近傍にＤｓを配置し、そのＤｓにＣｙ３−ｈｘ−ｄＰｘが相補して特異的に組み込まれると、３６５ｎｍ付近の照射でｓとＣｙ３間にＦＲＥＴが起こり、ＰＣＲ増幅された二本鎖ＤＮＡのみを特異的に光らせることができる。図２５の場合にはｓ塩基がプライマー中に２つ入るので、ＰＣＲによる相補鎖合成がこの部分で停止する可能性があるが、本法ではｓを天然型塩基にリンカーを介して結合しているので、ＰＣＲでの相補鎖合成が進む。これにより、人工塩基を含む発色のための部位をプライマー中のどの部分にも導入できＬＡＭＰ法やＳＭＡＰ法などのＰＣＲ法に利用できる。またパドロックＰＣＲ法などのプライマー領域以外にも本手法を用いることができる。 PCR was performed in the presence of a Cy3-hx-dPxTP substrate using a primer containing two adjacent modified bases (s-hx-dU) in which s, which is a fluorescent artificial base, was bound to a natural base U via a linker. The principle of real-time PCR when performing is shown in FIG. 29a. When two s-hx-dUs are introduced adjacently, the fluorescence of s is quenched as in the case where two s are introduced adjacently (FIG. 25). However, when Ds is arranged in the vicinity of s-hx-dU and Cy3-hx-dPx is specifically incorporated into the Ds in a complementary manner, FRET occurs between s and Cy3 by irradiation near 365 nm, and PCR amplification Only the double-stranded DNA thus prepared can be specifically illuminated. In the case of FIG. 25, two s bases are included in the primer, and thus complementary strand synthesis by PCR may stop at this part. In this method, s is bound to a natural base via a linker. Therefore, complementary strand synthesis by PCR proceeds. As a result, a site for color development containing an artificial base can be introduced into any part of the primer and can be used for PCR methods such as the LAMP method and the SMAP method. In addition, this technique can be used in addition to primer regions such as padlock PCR.
　図２９ｂは、用いたＤＮＡの配列、ならびにＰＣＲの条件を示した。図２９ｃには５５サイクルのリアルタイムＰＣＲの結果を、図２９ｄには５５サイクルのＰＣＲ後の増幅産物を目視で確認した結果をそれぞれ示す。標的とするＤＮＡ（ターゲットＤＮＡ）の量を０−３００００００コピーの間でＰＣＲ増幅を行ったところ、３コピー以上で目視判定ができた。 FIG. 29b shows the DNA sequence used and the PCR conditions. FIG. 29c shows the results of 55 cycles of real-time PCR, and FIG. 29d shows the results of visual confirmation of amplification products after 55 cycles of PCR. When PCR amplification was performed between 0 and 3000000 copies of target DNA (target DNA), visual determination was possible with 3 or more copies.
　実験に使用した配列（下線部はプライマーアニーリング部位に相当；Ｕｓ＝ｓ−ｈｘ−ｄＵ）
　５’−プライマー配列　：５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣ−３’（配列番号１５）
　３’−プライマー配列　：５’−　ＡＡＴＡＡＵｓＵｓＧＣＤｓＴＣＣＴＣＡＡＡＧＧＴＧＧＴＧＡＣＴＴＣ−３’（配列番号２７）
　二本鎖鋳型ＤＮＡ　（９８　ｂｐ；片方の鎖のみ記載）：
　５’−ＣＡＴＧＴＡＧＡＴＧＣＣＡＴＣＡＡＡＧＡＡＧＣＴＣＴＧＡＧＣＣＴＣＣＴＡＡＡＴＧＡＣＡＴＧＣＧＴＧＣＴＣＴＧＧＡＧＡＡＣＧＡＡＡＡＧＡＡＣＧＡＡＧＡＣＧＴＡＧＡＡＧＴＣＡＣＣＡＣＣＴＴＴＧＡＧＧＡ−３’（配列番号１７）
　具体的には、リアルタイムＰＣＲ装置（ストラタジーン、Ｍｘ３００５Ｐ）を用いて、プライマー各１μＭ、天然型塩基基質ｄＮＴＰ各０．２ｍＭ、人工塩基基質Ｃｙ３−ｈｘ−ｄＰｘＴＰ　２μＭの存在下、９４℃−２分の後、９４℃−５秒、６８℃−４０秒を１サイクルとする２ステップＰＣＲの条件で５５サイクルのＰＣＲを行った。ＰＣＲの反応スケールは２５μｌで、反応液の組成は４０ｍＭ　Ｔｒｉｃｉｎｅ−ＫＯＨ（ｐＨ８．０）、１６ｍＭ　ＫＣｌ、３．５ｍＭ　ＭｇＳＯ４、３．７５μｇ／ｍｌ　ＢＳＡ、１×ＴｉｔａｎｉｕｍＴａｑ　ＤＮＡポリメラーゼである。鋳型に用いたＤＮＡ断片は、反応液中にそれぞれ、０、３、３０、３００、３０００、３００００、３０００００、３００００００コピーになるように希釈し、それぞれの濃度につき、ＰＣＲを実施した。その反応チューブを直接、３６５ｎｍのＵＶで照射し、オレンジフィルターを通して目視で蛍光を検出した。 Sequence used for experiment (underlined portion corresponds to primer annealing site; Us = s-hx-dU)
5′-primer sequence: 5′- CATGTAGATGCCCATCAAAGAAGCTC- 3 ′ (SEQ ID NO: 15)
3'-primer sequence: 5'-AATAAUsGCDs TCCTCAAAGGTGGTGACTTC- 3 '(SEQ ID NO: 27)
Double-stranded template DNA (98 bp; only one strand is listed):
5′- CATGTAGATGCCCCATCAAAGAAGCTC TGAGCCCTCCTAAAATGACATGCGTGCTCTGGAGAACGAAAAGAAACGAAGACGTA GAAGTCACCACCTTTGAGGA- 3 ′ (SEQ ID NO: 17)
Specifically, using a real-time PCR apparatus (Stratagene, Mx3005P), 94 ° C.-2 minutes in the presence of each primer 1 μM, natural base substrate dNTP 0.2 mM, artificial base substrate Cy3-hx-dPxTP 2 μM Thereafter, 55 cycles of PCR were performed under the conditions of 2-step PCR with 94 ° C. for 5 seconds and 68 ° C. for 40 seconds as one cycle. The reaction scale of PCR is 25 μl, and the composition of the reaction solution is 40 mM Tricine-KOH (pH 8.0), 16 mM KCl, 3.5 mM MgSO 4, 3.75 μg / ml BSA, 1 × Titanium Taq DNA polymerase. The DNA fragment used for the template was diluted to 0, 3, 30, 300, 3000, 30000, 300000, 3000000 copies in the reaction solution, and PCR was performed for each concentration. The reaction tube was directly irradiated with 365 nm UV, and fluorescence was detected visually through an orange filter.
　実施例２１　ｓ−ｈｘ−ｄＵアミダイト試薬（図６の化合物）の化学合成（図３０）
　８−ブロモ−１−オクチンの合成（図３０の工程（ａ））
　８−ヒドロキシ−１−オクチン（１．９５ｇ，１５ｍｍｏｌ）に脱水ジクロロメタン（２０ｍｌ）、トリフェニルホスフィン（５．９１ｇ，２２．５ｍｍｏｌ）を加え、０℃に冷却後、脱水ジクロロメタン（１０ｍｌ）に溶解させた四臭化炭素（７．４６ｇ，２２．５ｍｍｏｌ）を滴下し、室温で２時間攪拌した。ジクロロメタン（１００ｍｌ）、５％　炭酸水素ナトリウム（１５０ｍｌ）で分液、有機層を飽和食塩水（１５０ｍｌ）で洗浄し、有機層を硫酸ナトリウムで乾燥後、濃縮した。これをシリカゲルカラムクロマトグラフィー（ジクロロメタン：メタノール＝１００：０→９９：１）で精製し、８−ブロモ−１−オクチン（ｃｒｕｄｅ）を得た。 Example 21 Chemical Synthesis of s-hx-dU Amidite Reagent (Compound of FIG. 6) (FIG. 30)
Synthesis of 8-bromo-1-octyne (step (a) in FIG. 30)
Dehydrated dichloromethane (20 ml) and triphenylphosphine (5.91 g, 22.5 mmol) were added to 8-hydroxy-1-octyne (1.95 g, 15 mmol), cooled to 0 ° C., and dissolved in dehydrated dichloromethane (10 ml). Carbon tetrabromide (7.46 g, 22.5 mmol) was added dropwise and stirred at room temperature for 2 hours. The mixture was separated with dichloromethane (100 ml), 5% sodium hydrogen carbonate (150 ml), the organic layer was washed with saturated brine (150 ml), and the organic layer was dried over sodium sulfate and concentrated. This was purified by silica gel column chromatography (dichloromethane: methanol = 100: 0 → 99: 1) to obtain 8-bromo-1-octyne (crude).
　８−ブロモ−１−オクチンの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　３．５１（ｔ，２Ｈ，Ｊ＝６．７Ｈｚ），２．７１（ｔ，１Ｈ，Ｊ＝２．７Ｈｚ），２．１２−２．１７（ｍ，２Ｈ），１．７５−１．８４（ｍ，２Ｈ），１．２４−１．５４（ｍ，６Ｈ）．
　２）　６−（チエン−２−イル）−９−（７−オクチニル）−２−アミノプリンの合成（図３０の工程（ｂ））
　６−（チエン−２−イル）−２−アミノプリン（１．２ｇ，５．５ｍｍｏｌ）と炭酸カリウム（２．３ｇ，１６．５ｍｍｏｌ）の脱水ジメチルホルムアミド（２５ｍｌ）溶液に、１）で得られた８−ブロモ−１−オクチン（２．０ｇ，１０．６ｍｍｏｌ）を加えて、室温で１５時間撹拌した。反応溶液を濃縮し、酢酸エチルと水で分液し、有機層を飽和食塩水で洗浄した。有機層を無水硫酸ナトリウムで乾燥し中圧分取カラムクロマトグラムにより精製して６−（チエン−２−イル）−９−（７−オクチニル）−２−アミノプリン（１．６ｇ，４．９ｍｍｏｌ，８７％）を得た。 Properties of 8-bromo-1 -octyne 1 H NMR (300 MHz, DMSO-d6) δ 3.51 (t, 2H, J = 6.7 Hz), 2.71 (t, 1H, J = 2.7 Hz) 2.12-2.17 (m, 2H), 1.75-1.84 (m, 2H), 1.24-1.54 (m, 6H).
2) Synthesis of 6- (thien-2-yl) -9- (7-octynyl) -2-aminopurine (step (b) of FIG. 30)
Obtained in 1) to a solution of 6- (thien-2-yl) -2-aminopurine (1.2 g, 5.5 mmol) and potassium carbonate (2.3 g, 16.5 mmol) in dehydrated dimethylformamide (25 ml) 8-Bromo-1-octyne (2.0 g, 10.6 mmol) was added and stirred at room temperature for 15 hours. The reaction solution was concentrated, partitioned between ethyl acetate and water, and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by medium pressure preparative column chromatogram to give 6- (thien-2-yl) -9- (7-octynyl) -2-aminopurine (1.6 g, 4.9 mmol). 87%).
　６−（チエン−２−イル）−９−（７−オクチニル）−２−アミノプリンの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　８．５３（ｄｄ，１Ｈ，Ｊ＝１．２，３．７Ｈｚ），８．１４（ｓ，１Ｈ），７．７９（ｄｄ，１Ｈ，Ｊ＝１．２，５．０Ｈｚ），７．２６（ｄｄ，１Ｈ，Ｊ＝３．７，５．０Ｈｚ），６．４８（ｂｒｓ，２Ｈ），４．０５（ｔ，２Ｈ，Ｊ＝７．２Ｈｚ），２．７２（ｔ，１Ｈ，Ｊ＝２．６Ｈｚ），２．１２（ｍ，２Ｈ），１．７８（ｍ，２Ｈ），１．２３−１．４６（ｍ，６Ｈ）．
　３）　６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリンの合成（図３０の工程（ｃ））
　１−ヒドロキシベンゾトリアゾール（１．１９ｇ，８．８４ｍｍｏｌ）を脱水ピリジンで３回共沸乾燥し、脱水ピリジン（２．５ｍｌ）、脱水アセトニトリル（２．５ｍｌ）、フェノキシアセチルクロリド（１．０８ｍｌ，７．８５ｍｍｏｌ）を加え、室温で５分間攪拌後、０℃に冷却し、これに脱水ピリジン（２５ｍＬ）に溶解した。２）で得られた６−（チエン−２−イル）−９−（７−オクチニル）−２−アミノプリン（１．６０ｇ，４．９１ｍｍｏｌ）を加え、室温で一晩攪拌した。酢酸エチル（１５０ｍｌ）、飽和食塩水（１５０ｍｌ×２回）で分液し、有機層を硫酸ナトリウムで乾燥後、濃縮した。これをシリカゲルカラムクロマトグラフィー（ジクロロメタン：メタノール＝１００：０→９９：１）で精製し、６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン（１．４４ｇ，３．１３ｍｍｏｌ，６４％）を得た。 Properties of 6- (thien-2-yl) -9- (7-octynyl) -2-aminopurine 1 H NMR (300 MHz, DMSO-d6) δ 8.53 (dd, 1H, J = 1.2, 3.7 Hz), 8.14 (s, 1 H), 7.79 (dd, 1 H, J = 1.2, 5.0 Hz), 7.26 (dd, 1 H, J = 3.7, 5.0 Hz) ), 6.48 (brs, 2H), 4.05 (t, 2H, J = 7.2 Hz), 2.72 (t, 1H, J = 2.6 Hz), 2.12 (m, 2H), 1.78 (m, 2H), 1.23-1.46 (m, 6H).
3) Synthesis of 6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidopurine (step (c) in FIG. 30)
1-Hydroxybenzotriazole (1.19 g, 8.84 mmol) was azeotropically dried three times with dehydrated pyridine, dehydrated pyridine (2.5 ml), dehydrated acetonitrile (2.5 ml), phenoxyacetyl chloride (1.08 ml, 7 .85 mmol) was added, stirred at room temperature for 5 minutes, cooled to 0 ° C., and dissolved in dehydrated pyridine (25 mL). 6- (thien-2-yl) -9- (7-octynyl) -2-aminopurine (1.60 g, 4.91 mmol) obtained in 2) was added and stirred at room temperature overnight. The mixture was partitioned between ethyl acetate (150 ml) and saturated brine (150 ml × twice), and the organic layer was dried over sodium sulfate and concentrated. This was purified by silica gel column chromatography (dichloromethane: methanol = 100: 0 → 99: 1) to give 6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidepurine (1 .44 g, 3.13 mmol, 64%).
　６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリンの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１０．７１（ｓ，１Ｈ），８
．６２（ｄ，１Ｈ，Ｊ＝２．６Ｈｚ），８．５４（ｓ，１Ｈ），７．９２（ｄｄ，１Ｈ，Ｊ＝１．１，５．０Ｈｚ），７．３１（ｍ，３Ｈ），６．９２−６．９３（ｍ，３Ｈ），５．１５（ｂｒｓ，２Ｈ），４．２０（ｔ，２Ｈ，Ｊ＝７．１Ｈｚ），２．７１（ｔ，１Ｈ，Ｊ＝２．６Ｈｚ），２．０９−２．１３（ｍ，２Ｈ），１．８２−１．９２（ｍ，２Ｈ），１．２７−１．４１（ｍ，６Ｈ）．
　４）　５−［６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジンの合成（図３０の工程（ｄ））
　５’−Ｏ−（４，４’−ジメトキシトリチル）−５−ヨード−２’−デオキシウリジン（１．６４ｇ，２．５ｍｍｏｌ）、テトラキス（トリフェニルホスフィン）パラジウム（０）（１４５ｍｇ，０．１２５ｍｍｏｌ）、ヨウ化銅（７６ｍｇ，０．４ｍｍｏｌ）、脱水ジメチルホルムアミド（７．５ｍｌ）を加え、アルゴンガスで置換し、脱水トリエチルアミン（５２３μｌ，３．７５ｍｍｏｌ）を加えた後、脱水ジメチルホルムアミド（５ｍｌ）、脱水ピリジン（１０ｍｌ）に溶解した、３）で得られた６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン（１．３８ｇ，３．００ｍｍｏｌ）を加え、マイクロウェーブ装置（スタンダードモード）で６０℃，３時間攪拌した。酢酸エチル（１００ｍｌ）、水（１００ｍｌ）で分液、有機層を飽和食塩水（１００ｍｌ）で洗浄し、有機層を硫酸ナトリウムで乾燥後、濃縮した。これをシリカゲルカラムクロマトグラフィー（ジクロロメタン：メタノール＝１００：０→９７：３）で精製し、５−［６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジン（９３１ｍｇ，０．９４ｍｍｏｌ，３８％）を得た。 Properties of 6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidopurine 1 H NMR (300 MHz, DMSO-d6) δ 10.71 (s, 1H), 8
. 62 (d, 1H, J = 2.6 Hz), 8.54 (s, 1H), 7.92 (dd, 1H, J = 1.1, 5.0 Hz), 7.31 (m, 3H), 6.92-6.93 (m, 3H), 5.15 (brs, 2H), 4.20 (t, 2H, J = 7.1 Hz), 2.71 (t, 1H, J = 2.6 Hz) ), 2.09-2.13 (m, 2H), 1.82-1.92 (m, 2H), 1.27-1.41 (m, 6H).
4) 5- [6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidepurine] -5′-O- (4,4′-dimethoxytrityl) -2′- Synthesis of deoxyuridine (step (d) in FIG. 30)
5′-O- (4,4′-dimethoxytrityl) -5-iodo-2′-deoxyuridine (1.64 g, 2.5 mmol), tetrakis (triphenylphosphine) palladium (0) (145 mg, 0.125 mmol) ), Copper iodide (76 mg, 0.4 mmol) and dehydrated dimethylformamide (7.5 ml) were added, and replaced with argon gas, dehydrated triethylamine (523 μl, 3.75 mmol) was added, and dehydrated dimethylformamide (5 ml) 6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidopurine (1.38 g, 3.00 mmol) obtained in 3) dissolved in dehydrated pyridine (10 ml) And stirred with a microwave apparatus (standard mode) at 60 ° C. for 3 hours. The mixture was separated with ethyl acetate (100 ml) and water (100 ml), the organic layer was washed with saturated brine (100 ml), and the organic layer was dried over sodium sulfate and concentrated. This was purified by silica gel column chromatography (dichloromethane: methanol = 100: 0 → 97: 3) to give 5- [6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamide. Purine] -5′-O- (4,4′-dimethoxytrityl) -2′-deoxyuridine (931 mg, 0.94 mmol, 38%) was obtained.
　５−［６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジンの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１１．５９（ｂｒｓ，１Ｈ），
　１０．７０（ｂｒｓ，１Ｈ），８．６１（ｄｄ，１Ｈ，Ｊ＝０．９，３．８Ｈｚ），８．５１（ｓ，１Ｈ），７．９２（ｄｄ，１Ｈ，Ｊ＝０．９，５．０Ｈｚ），７．８７（ｓ，１Ｈ），７．１７−７．３７（ｍ，１２Ｈ），６．８２−６．９６（ｍ，７Ｈ），６．１１（ｔ，１Ｈ，Ｊ＝６．６Ｈｚ），５．３１（ｄ，１Ｈ，Ｊ＝４．４Ｈｚ），５．１４（ｂｒｓ，２Ｈ），４．０２−４．２８（ｍ，３Ｈ），３．７０−３．９１（ｍ，１Ｈ），３．１２−３．１６（ｍ，２Ｈ），２．０４−２．２４（ｍ，４Ｈ），１．７６−１．９９（ｍ，２Ｈ），１．１５−１．２０（ｍ，６Ｈ）．
　５）　５−［６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジン−３’−Ｏ−（２−シアノエチル−Ｎ，Ｎ−ジイソプロピル）ホスホロアミダイトの合成（図３０の工程（ｅ））
　４）で得られた５−［６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジン（８９０ｍｇ，０．９ｍｍｏｌ）を脱水ピリジンで３回、脱水テトラヒドロフランで３回共沸乾燥した。次いで、脱水テトラヒドロフラン（４．５ｍｌ）、脱水ジイソプロピルエチルアミン（２３５μｌ，１．３５ｍｍｏｌ）、２−シアノエチル　Ｎ，Ｎ’−ジイソプロピルクロロホスホルアミジド（２４１μｌ，１．０８ｍｍｏｌ）を加え、室温で１時間攪拌した。脱水メタノール（５０μｌ）を加え、酢酸エチル：トリエチルアミン（２０：１，５０ｍｌ）、５％　炭酸水素ナトリウム（５０ｍｌ）で分液、有機層を飽和食塩水（１００ｍｌ）で洗浄し、有機層を硫酸ナトリウムで乾燥後、濃縮した。これをシリカゲルカラムクロマトグラフィー（ヘキサン：酢酸エチル：トリエチルアミン＝９８：０：２→７８：２０：２）で精製し、５−［６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン］−５’−Ｏ−（４，４’−ジメトキシ
トリチル）−２’−デオキシウリジン−３’−Ｏ−（２−シアノエチル−Ｎ，Ｎ−ジイソプロピル）ホスホロアミダイト（８６７ｍｇ，０．７３ｍｍｏｌ，８１％）を得た。 5- [6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidepurine] -5′-O- (4,4′-dimethoxytrityl) -2′-deoxyuridine physical properties 1 H NMR (300MHz, DMSO- d6) δ 11.59 (brs, 1H),
10.70 (brs, 1H), 8.61 (dd, 1H, J = 0.9, 3.8 Hz), 8.51 (s, 1H), 7.92 (dd, 1H, J = 0.9) , 5.0 Hz), 7.87 (s, 1H), 7.17-7.37 (m, 12H), 6.82-6.96 (m, 7H), 6.11 (t, 1H, J = 6.6 Hz), 5.31 (d, 1H, J = 4.4 Hz), 5.14 (brs, 2H), 4.02-4.28 (m, 3H), 3.70-3.91. (M, 1H), 3.12-3.16 (m, 2H), 2.04-2.24 (m, 4H), 1.76-1.99 (m, 2H), 1.15-1 20 (m, 6H).
5) 5- [6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidepurine] -5′-O- (4,4′-dimethoxytrityl) -2′- Synthesis of deoxyuridine-3′-O- (2-cyanoethyl-N, N-diisopropyl) phosphoramidite (step (e) in FIG. 30)
5- [6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidopurine] -5′-O- (4,4′-dimethoxytrityl) obtained in 4) -2'-deoxyuridine (890 mg, 0.9 mmol) was azeotropically dried three times with dehydrated pyridine and three times with dehydrated tetrahydrofuran. Next, dehydrated tetrahydrofuran (4.5 ml), dehydrated diisopropylethylamine (235 μl, 1.35 mmol) and 2-cyanoethyl N, N′-diisopropylchlorophosphoramidide (241 μl, 1.08 mmol) were added and stirred at room temperature for 1 hour. did. Add dehydrated methanol (50 μl), separate with ethyl acetate: triethylamine (20: 1, 50 ml), 5% sodium bicarbonate (50 ml), wash the organic layer with saturated brine (100 ml), and wash the organic layer with sodium sulfate. After drying, it was concentrated. This was purified by silica gel column chromatography (hexane: ethyl acetate: triethylamine = 98: 0: 2 → 78: 20: 2) to give 5- [6- (thien-2-yl) -9- (7-octynyl). -2-phenoxyacetamidepurine] -5′-O- (4,4′-dimethoxytrityl) -2′-deoxyuridine-3′-O- (2-cyanoethyl-N, N-diisopropyl) phosphoramidite (867 mg, 0.73 mmol, 81%) was obtained.
　５−［６−（チエン−２−イル）−９−（７−オクチニル）−２−フェノキシアセタミドプリン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジン−３’−Ｏ−（２−シアノエチル−Ｎ，Ｎ−ジイソプロピル）ホスホロアミダイトの物性値 １Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１１．５７（ｂｒｓ，１Ｈ），１０．７０（ｂｒｓ，１Ｈ），８．６０（ｄｄ，１Ｈ，Ｊ＝１．１，３．７Ｈｚ），８．５０（ｓ，１Ｈ），７．８９−７．９２（ｍ，２Ｈ），７．１４−７．３６（ｍ，１２Ｈ），６．７９−６．９５（ｍ，７Ｈ），６．１０（ｄｔ，１Ｈ，Ｊ＝６．２，６．３Ｈｚ），５．１３（ｂｒｓ，２Ｈ），４．５０−４．６０（ｍ，１Ｈ），４．１６（ｔ，２Ｈ，Ｊ＝６．７Ｈｚ），３．９９−４．０６（ｍ，１Ｈ），３．１７−３．７１（ｍ，１２Ｈ），２．２６−２．７６（ｍ，４Ｈ），２．０５−２．１０（ｍ，２Ｈ），１．７４−１．７７（ｍ，２Ｈ），０．８２−１．３９（ｍ，１８Ｈ）．
　３１Ｐ　ＮＭＲ（１２１ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１４８．６７，１４８．３２．
　実施例２２　Ｄｓｓ−ｈｘ−ｄＵアミダイト試薬（図６の化合物）の化学合成（図３３）
１）７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジンの合成（図３３の工程（ａ））
　７−（２，２’−ビチエン−５−イル）−イミダゾ［４，５−ｂ］ピリジン（８５０ｍｇ，３．０ｍｍｏｌ）と炭酸カリウム（１．３ｇ，９．０ｍｍｏｌ）のＤＭＦ（１５ｍｌ）溶液を６０℃で１時間撹拌した。次いで、８−ブロモ−１−オクチン（８５０ｍｇ，４．５ｍｍｏｌ）を加えて、６０℃で６時間撹拌した。反応溶液を酢酸エチルと水で分液し、有機層を飽和食塩水で洗浄した。有機層を無水硫酸ナトリウムで乾燥し中圧分取カラムクロマトグラムにより精製して７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン（５２０ｍｇ，１．３ｍｍｏｌ，４４％）を得た。 5- [6- (thien-2-yl) -9- (7-octynyl) -2-phenoxyacetamidepurine] -5′-O- (4,4′-dimethoxytrityl) -2′-deoxyuridine Properties of -3′-O- (2-cyanoethyl-N, N-diisopropyl) phosphoramidite 1 H NMR (300 MHz, DMSO-d6) δ 11.57 (brs, 1H), 10.70 (brs, 1H ), 8.60 (dd, 1H, J = 1.1, 3.7 Hz), 8.50 (s, 1H), 7.89-7.92 (m, 2H), 7.14-7.36. (M, 12H), 6.79-6.95 (m, 7H), 6.10 (dt, 1H, J = 6.2, 6.3 Hz), 5.13 (brs, 2H), 4.50 -4.60 (m, 1H), 4.16 (t, 2H, J = 6.7 Hz), 3.99-4.06 ( m, 1H), 3.17-3.71 (m, 12H), 2.26-2.76 (m, 4H), 2.05-2.10 (m, 2H), 1.74-1. 77 (m, 2H), 0.82-1.39 (m, 18H).
31 P NMR (121 MHz, DMSO-d6) δ 148.67, 148.32.
Example 22 Chemical synthesis of Dss-hx-dU amidite reagent (compound of FIG. 6) (FIG. 33)
1) Synthesis of 7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine (step (a) in FIG. 33)
A solution of 7- (2,2′-bitien-5-yl) -imidazo [4,5-b] pyridine (850 mg, 3.0 mmol) and potassium carbonate (1.3 g, 9.0 mmol) in DMF (15 ml) was added. Stir at 60 ° C. for 1 hour. Subsequently, 8-bromo-1-octyne (850 mg, 4.5 mmol) was added, and the mixture was stirred at 60 ° C. for 6 hours. The reaction solution was partitioned between ethyl acetate and water, and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by medium pressure preparative column chromatogram to obtain 7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b. ] Pyridine (520 mg, 1.3 mmol, 44%) was obtained.
　７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジンの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　８．５６（ｓ，１Ｈ），８．３４（ｄ，１Ｈ，Ｊ＝５．２Ｈｚ），８．２１（ｄ，１Ｈ，Ｊ＝３．９Ｈｚ），７．６３（ｄ，１Ｈ，Ｊ＝５．２Ｈｚ），７．５８（ｄｄ，１Ｈ，Ｊ＝１．１，５．１Ｈｚ），７．４６（ｄｄ，１Ｈ，Ｊ＝１．１，３．６Ｈｚ），７．４４（ｄ，１Ｈ，Ｊ＝４．０Ｈｚ），７．１４（ｄｄ，１Ｈ，Ｊ＝３．６，５．１Ｈｚ），４．２９（ｔ，２Ｈ，Ｊ＝７．４Ｈｚ），２．７２（ｔ，１Ｈ，Ｊ＝２．７Ｈｚ），２．１２（ｍ，２Ｈ），１．８７（ｍ，２Ｈ），１．４３−１．３１（ｍ，６Ｈ）．
　２）　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−２’−デオキシウリジンの合成（図３３の工程（ｂ））
　５−ヨード−２’−デオキシウリジン（２９４ｍｇ，０．８３ｍｍｏｌ）、７−（２，２’−ビチエニル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン（２７０ｍｇ，０．６９ｍｍｏｌ）、ＣｕＩ（２５ｍｇ）、テトラキストリフェニルホスフィン（４８ｍｇ）、トリエチルアミン（１７３μｌ）のＤＭＦ（４．２ｍｌ）溶液を室温で１７時間撹拌した。反応溶液を酢酸エチルと水で分液し、有機層を飽和食塩水で洗浄した。有機層を無水硫酸ナトリウムで乾燥しカラムクロマトグラム（３％メタノールの塩化メチレン溶液で溶出）により精製して５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−２’−デオキシウリジン（１５５ｍｇ，０．２５ｍｍｏｌ，３６％）を得た。 Physical property value of 7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine 1 H NMR (300 MHz, DMSO-d6) δ 8.56 ( s, 1H), 8.34 (d, 1H, J = 5.2 Hz), 8.21 (d, 1H, J = 3.9 Hz), 7.63 (d, 1H, J = 5.2 Hz), 7.58 (dd, 1H, J = 1.1, 5.1 Hz), 7.46 (dd, 1H, J = 1.1, 3.6 Hz), 7.44 (d, 1H, J = 4. 0 Hz), 7.14 (dd, 1 H, J = 3.6, 5.1 Hz), 4.29 (t, 2 H, J = 7.4 Hz), 2.72 (t, 1 H, J = 2.7 Hz) ), 2.12 (m, 2H), 1.87 (m, 2H), 1.43-1.31 (m, 6H).
2) Synthesis of 5- [7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -2′-deoxyuridine (FIG. 33) Step (b))
5-Iodo-2′-deoxyuridine (294 mg, 0.83 mmol), 7- (2,2′-bithienyl) -3- (7-octynyl) -imidazo [4,5-b] pyridine (270 mg, 0. 0). 69 mmol), CuI (25 mg), tetrakistriphenylphosphine (48 mg), triethylamine (173 μl) in DMF (4.2 ml) was stirred at room temperature for 17 hours. The reaction solution was partitioned between ethyl acetate and water, and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by column chromatogram (eluted with 3% methanol in methylene chloride) to give 5- [7- (2,2′-bithien-5-yl) -3- (7- Octynyl) -imidazo [4,5-b] pyridine] -2′-deoxyuridine (155 mg, 0.25 mmol, 36%) was obtained.
　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−２’−デオキシウリジンの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１１．５４（ｓ，１Ｈ），８．５６（ｓ，１Ｈ），８．３４（ｄ，１Ｈ，Ｊ＝５．２Ｈｚ），８．２１（ｄ，１Ｈ，Ｊ＝３．９Ｈｚ），８．０９（ｓ，１Ｈ），７．６３（ｄ，１Ｈ，Ｊ＝５．２Ｈｚ），７．５８（ｄｄ，１Ｈ，Ｊ＝１．１，５．１Ｈｚ），７．４６（ｄｄ，１Ｈ，Ｊ＝１．１，３．６Ｈｚ），７．４４（ｄ，１Ｈ，Ｊ＝４．１Ｈｚ），７．１４（ｄｄ，１Ｈ，Ｊ＝３．６，５．１Ｈｚ），６．１０（ｔ，１Ｈ，Ｊ＝６．９Ｈｚ），５．２１（ｄ，１Ｈ，Ｊ＝４．３Ｈｚ），５．０６（ｔ，１Ｈ，Ｊ＝５．０Ｈｚ），４．３０（ｔ，２Ｈ，Ｊ＝７．２Ｈｚ），４．２１（ｍ，１Ｈ），３．７７（ｍ，１Ｈ），３．５６（ｍ，２Ｈ），２．３３（ｍ，２Ｈ），２．０９（ｍ，２Ｈ），１．８８（ｍ，２Ｈ），１．４５（ｍ，４Ｈ），１．２９（ｍ，２Ｈ）．
　３）　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−５’−Ｏ−（４，４−ジメトキシトリチル）−２’−デオキシウリジンの合成（図３３の工程（ｃ））
　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−２’−デオキシウリジン（１５０ｍｇ，０．２４ｍｍｏｌ）、４，４’−ジメトキシトリチルクロリド（９１ｍｇ，０．２７ｍｍｏｌ）のピリジン（２．４ｍｌ）溶液を室温で１時間撹拌した。反応溶液を酢酸エチルと５％炭酸水素ナトリウム水溶液で分液し、有機層を飽和食塩水で洗浄した。有機層を無水硫酸ナトリウムで乾燥しカラムクロマトグラム（２％メタノールの塩化メチレン溶液で溶出）により精製して５−［７−（２，２’−ビチエニル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−５’−Ｏ−（４，４−ジメトキシトリチル）−２’−デオキシウリジン（１８３ｍｇ，０．２ｍｍｏｌ，８２％）を得た。 Properties of 5- [7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -2′-deoxyuridine 1 H NMR (300 MHz , DMSO-d6) δ 11.54 (s, 1H), 8.56 (s, 1H), 8.34 (d, 1H, J = 5.2 Hz), 8.21 (d, 1H, J = 3) 0.9 Hz), 8.09 (s, 1 H), 7.63 (d, 1 H, J = 5.2 Hz), 7.58 (dd, 1 H, J = 1.1, 5.1 Hz), 7.46. (Dd, 1H, J = 1.1, 3.6 Hz), 7.44 (d, 1H, J = 4.1 Hz), 7.14 (dd, 1H, J = 3.6, 5.1 Hz), 6.10 (t, 1H, J = 6.9 Hz), 5.21 (d, 1H, J = 4.3 Hz), 5.06 (t, 1H, J = 5.0 Hz), 4.30 (t 2H, J = 7.2 Hz), 4.21 (m, 1H), 3.77 (m, 1H), 3.56 (m, 2H), 2.33 (m, 2H), 2.09 (m , 2H), 1.88 (m, 2H), 1.45 (m, 4H), 1.29 (m, 2H).
3) 5- [7- (2,2′-Bitien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -5′-O- (4,4-dimethoxy Synthesis of trityl) -2′-deoxyuridine (step (c) in FIG. 33)
5- [7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -2′-deoxyuridine (150 mg, 0.24 mmol), A solution of 4,4′-dimethoxytrityl chloride (91 mg, 0.27 mmol) in pyridine (2.4 ml) was stirred at room temperature for 1 hour. The reaction solution was partitioned between ethyl acetate and 5% aqueous sodium hydrogen carbonate solution, and the organic layer was washed with saturated brine. The organic layer was dried over anhydrous sodium sulfate and purified by column chromatogram (eluted with 2% methanol in methylene chloride) to give 5- [7- (2,2′-bithienyl) -3- (7-octynyl) -imidazo. [4,5-b] pyridine] -5′-O- (4,4-dimethoxytrityl) -2′-deoxyuridine (183 mg, 0.2 mmol, 82%) was obtained.
　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−５’−Ｏ−（４，４−ジメトキシトリチル）−２’−デオキシウリジンの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１１．５８（ｓ，１Ｈ），８．５３（ｓ，１Ｈ），８．３２（ｄ，１Ｈ，Ｊ＝５．２Ｈｚ），８．２０（ｄ，１Ｈ，Ｊ＝３．９Ｈｚ），７．８７（ｓ，１Ｈ），７．６０−７．５７（ｍ，２Ｈ），７．４６−７．４３（ｍ，２Ｈ），７．３５−７．３２（ｍ，２Ｈ），７．２６−７．１３（ｍ，８Ｈ），６．８１（ｄ，４Ｈ，Ｊ＝９．０Ｈｚ），６．１０（ｔ，１Ｈ，Ｊ＝７．０Ｈｚ），５．３０（ｄ，１Ｈ，Ｊ＝４．４Ｈｚ），４．２６（ｍ，３Ｈ），３．８９（ｍ，１Ｈ），３．６９（ｓ，６Ｈ），３．１５（ｍ，２Ｈ），２．１８（ｍ，２Ｈ），２．０５（ｍ，２Ｈ），１．７８（ｍ，２Ｈ），１．２２−１．１３（ｍ，６Ｈ）．
　４）　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジン−３’−Ｏ−（２−シアノエチル−Ｎ，Ｎ−ジイソプロピル）ホスホロアミダイトの合成（図３３の工程（ｄ））
　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−５’−Ｏ−（４，４−ジメトキシトリチル）−２’−デオキシウリジン　（１８０ｍｇ，０．２ｍｍｏｌ）をピリジンで３回、ＴＨＦで３回共沸乾燥した後、ＴＨＦ（１．０ｍｌ）とジイソプロピルエチルアミン（５２μｌ）を加えて撹拌した。この溶液に、２−シアノエチル−Ｎ，Ｎ−ジイソプロピルクロロホスホロアミダイト（５４μｌ，０．２４ｍｍｏｌ）を加えて室温で１時間撹拌した。反応溶液に脱水メタノール（５０μｌ）を加えた後、酢酸エチル／トリエチルアミン（２０：１，ｖ／ｖ）と５％炭酸水素ナトリウム水溶液で分液した。有機層を飽和食塩水で洗浄し、無水硫酸ナトリウムで乾燥して濃縮した。残さをシリカゲルカラムクロマトグラム（酢酸エチル：塩化メチレン：トリエチルアミン、４５：４５：１０、ｖ／ｖ／ｖで溶出）で精製して５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジン−３’−Ｏ−（２−シアノエチル−Ｎ，Ｎ−ジイソプロピル）ホスホロアミダイト（２２０ｍｇ，９９％）を得た。 5- [7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -5′-O- (4,4-dimethoxytrityl) Properties of -2'-deoxyuridine 1 H NMR (300 MHz, DMSO-d6) δ 11.58 (s, 1H), 8.53 (s, 1H), 8.32 (d, 1H, J = 5. 2Hz), 8.20 (d, 1H, J = 3.9 Hz), 7.87 (s, 1H), 7.60-7.57 (m, 2H), 7.46-7.43 (m, 2H), 7.35-7.32 (m, 2H), 7.26-7.13 (m, 8H), 6.81 (d, 4H, J = 9.0 Hz), 6.10 (t, 1H, J = 7.0 Hz), 5.30 (d, 1H, J = 4.4 Hz), 4.26 (m, 3H), 3.89 (m, 1H), 3.69 (s, 6H) 3.15 (m, 2H), 2.18 (m, 2H), 2.05 (m, 2H), 1.78 (m, 2H), 1.22-1.13 (m, 6H).
4) 5- [7- (2,2′-Bitien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -5′-O— (4,4′- Synthesis of dimethoxytrityl) -2′-deoxyuridine-3′-O- (2-cyanoethyl-N, N-diisopropyl) phosphoramidite (step (d) in FIG. 33)
5- [7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -5′-O- (4,4-dimethoxytrityl) -2′-deoxyuridine (180 mg, 0.2 mmol) was azeotropically dried three times with pyridine and three times with THF, and then THF (1.0 ml) and diisopropylethylamine (52 μl) were added and stirred. To this solution, 2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (54 μl, 0.24 mmol) was added and stirred at room temperature for 1 hour. Dehydrated methanol (50 μl) was added to the reaction solution, and the mixture was partitioned between ethyl acetate / triethylamine (20: 1, v / v) and 5% aqueous sodium hydrogen carbonate solution. The organic layer was washed with saturated brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by silica gel column chromatogram (ethyl acetate: methylene chloride: triethylamine, 45:45:10, eluted with v / v / v) to give 5- [7- (2,2′-bitien-5-yl). -3- (7-octynyl) -imidazo [4,5-b] pyridine] -5'-O- (4,4'-dimethoxytrityl) -2'-deoxyuridine-3'-O- (2-cyanoethyl -N, N-diisopropyl) phosphoramidite (220 mg, 99%) was obtained.
　５−［７−（２，２’−ビチエン−５−イル）−３−（７−オクチニル）−イミダゾ［４，５−ｂ］ピリジン］−５’−Ｏ−（４，４’−ジメトキシトリチル）−２’−デオキシウリジン−３’−Ｏ−（２−シアノエチル−Ｎ，Ｎ−ジイソプロピル）ホスホロアミダイトの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１１．５９（ｓ，１Ｈ），８．５３（ｓ，ｓ，１Ｈ，１Ｈ），８．３２（ｄ，１Ｈ，Ｊ＝５．２Ｈｚ），８．２０（ｄ，１Ｈ，Ｊ＝３．９Ｈｚ），７．８９（ｄ，１Ｈ，Ｊ＝２．１Ｈｚ），７．６０−７．５７（ｍ，２Ｈ），７．４６−７．４３（ｍ，２Ｈ），７．３４（ｍ，２Ｈ），７．２６−７．１３（ｍ，８Ｈ），６．８１（ｍ，４Ｈ），６．９８（ｄｔ，１Ｈ，Ｊ＝６．３，６．５Ｈｚ），４．４７（ｍ，１Ｈ），４．２５（ｔ，２Ｈ，Ｊ＝６．９Ｈｚ），４．０５−３．９８（ｍ，１Ｈ），３．７１（ｍ，１Ｈ），３．６９（ｓ，６Ｈ），３．６０−３．４２（ｍ，２Ｈ），３．２０（ｍ，２Ｈ），２．７３（ｔ，１Ｈ，Ｊ＝５．９Ｈｚ），２．６１（ｔ，１Ｈ，Ｊ＝５．９Ｈｚ），２．４４−２．２５（ｍ，２Ｈ），２．０７（ｍ，２Ｈ），１．７７（ｍ，２Ｈ），１．０９（ｍ，１８Ｈ）．
　３１Ｐ　ＮＭＲ（１２１ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　１４８．６８，１４８．３２．
　実施例２３　図２及び図３の化合物の合成 5- [7- (2,2′-bithien-5-yl) -3- (7-octynyl) -imidazo [4,5-b] pyridine] -5′-O- (4,4′-dimethoxytrityl) ) -2'-deoxyuridine-3'-O- (2-cyanoethyl-N, N-diisopropyl) phosphoramidite physical properties 1 H NMR (300 MHz, DMSO-d6) δ 11.59 (s, 1H), 8.53 (s, s, 1H, 1H), 8.32 (d, 1H, J = 5.2 Hz), 8.20 (d, 1H, J = 3.9 Hz), 7.89 (d, 1H) , J = 2.1 Hz), 7.60-7.57 (m, 2H), 7.46-7.43 (m, 2H), 7.34 (m, 2H), 7.26-7.13. (M, 8H), 6.81 (m, 4H), 6.98 (dt, 1H, J = 6.3, 6.5 Hz), 4.47 (m, 1H) 4.25 (t, 2H, J = 6.9 Hz), 4.05-3.98 (m, 1H), 3.71 (m, 1H), 3.69 (s, 6H), 3.60. -3.42 (m, 2H), 3.20 (m, 2H), 2.73 (t, 1H, J = 5.9 Hz), 2.61 (t, 1H, J = 5.9 Hz), 2 .44-2.25 (m, 2H), 2.07 (m, 2H), 1.77 (m, 2H), 1.09 (m, 18H).
31 P NMR (121 MHz, DMSO-d6) δ 148.68, 148.32.
Example 23 Synthesis of compounds of FIGS. 2 and 3
Figure JPOXMLDOC01-appb-C000055
Figure JPOXMLDOC01-appb-C000055
　１）　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロールの合成
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−２−ニトロピロール（４５６ｍｇ，２ｍｍｏｌ）のアセトニトリル（８ｍｌ）溶液にＮ−ヨードスクシンイミド（９００ｍｇ，４ｍｍｏｌ）を加えた。室温で一晩反応後、酢酸エチル（２００ｍｌ）、水（２００ｍｌ）で分液し、有機層を濃縮した。これをシリカゲルカラムクロマトグラフィーおよびＨＰＬＣ精製し、１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロール（５８７ｍｇ，１．６６ｍｍｏｌ，８３％）を得た。 1) Synthesis of 1- (2-deoxy-β-D-ribofuranosyl) -4-iodo-2-nitropyrrole 1- (2-deoxy-β-D-ribofuranosyl) -2-nitropyrrole (456 mg, 2 mmol) N-iodosuccinimide (900 mg, 4 mmol) was added to an acetonitrile (8 ml) solution. After reacting overnight at room temperature, the mixture was partitioned between ethyl acetate (200 ml) and water (200 ml), and the organic layer was concentrated. This was purified by silica gel column chromatography and HPLC to give 1- (2-deoxy-β-D-ribofuranosyl) -4-iodo-2-nitropyrrole (587 mg, 1.66 mmol, 83%).
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロールの物性値
　１Ｈ　ＮＭＲ（２７０ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　７．９０（ｄ，１Ｈ，Ｊ＝２．０Ｈｚ），７．４０（ｄ，１Ｈ，Ｊ＝２．０Ｈｚ），６．５４（ｔ，１Ｈ，Ｊ＝５．６Ｈｚ），５．２７（ｄ，１Ｈ，Ｊ＝４．３Ｈｚ），５．１０（ｔ，１Ｈ，Ｊ＝４．９Ｈｚ），４．２３（ｍ，１Ｈ），３．８３（ｍ，１Ｈ），３．５３−３．８５（ｍ，２Ｈ），２．１８−２．４５（ｍ，２Ｈ）．
　２）　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（チエン−２−イル）−２−ニトロピロールの合成
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロール（１７７ｍｇ，０．５ｍｍｏｌ）、二塩化ビス（トリフェニルホスフィン）パラジウム（ＩＩ）（１８ｍｇ，０．０２５ｍｍｏｌ）のＤＭＦ（２．５ｍｌ）溶液に２−（トリブチルスタニル）チオフェン（４７６μｌ，１．５ｍｍｏｌ）を加えた。マイクロウェーブ装置（スタンダードモード）で１００℃，３０分間反応後、酢酸エチル（５０ｍｌ）、水（５０ｍｌ）で分液し、有機層を濃縮した。これをＨＰＬＣ精製し、１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（チエン−２−イル）−２−ニトロピロール（９７ｍｇ，０．３２ｍｍｏｌ，６３％）を得た。 1- (2-deoxy-β-D-ribofuranosyl) -4-iodo-2-nitropyrrole property value 1 H NMR (270 MHz, DMSO-d6) δ 7.90 (d, 1H, J = 2.0 Hz) 7.40 (d, 1H, J = 2.0 Hz), 6.54 (t, 1H, J = 5.6 Hz), 5.27 (d, 1H, J = 4.3 Hz), 5.10 ( t, 1H, J = 4.9 Hz), 4.23 (m, 1H), 3.83 (m, 1H), 3.53-3.85 (m, 2H), 2.18-2.45 ( m, 2H).
2) Synthesis of 1- (2-deoxy-β-D-ribofuranosyl) -4- (thien-2-yl) -2-nitropyrrole 1- (2-deoxy-β-D-ribofuranosyl) -4-iodo- To a solution of 2-nitropyrrole (177 mg, 0.5 mmol), bis (triphenylphosphine) palladium dichloride (II) (18 mg, 0.025 mmol) in DMF (2.5 ml) was added 2- (tributylstannyl) thiophene (476 μl). , 1.5 mmol). After reacting at 100 ° C. for 30 minutes in a microwave apparatus (standard mode), the mixture was separated with ethyl acetate (50 ml) and water (50 ml), and the organic layer was concentrated. This was purified by HPLC to obtain 1- (2-deoxy-β-D-ribofuranosyl) -4- (thien-2-yl) -2-nitropyrrole (97 mg, 0.32 mmol, 63%).
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（チエン−２−イル）−２−ニトロピロールの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　８．１３（ｄ，１Ｈ，Ｊ＝２．３Ｈｚ），７．５２（ｄ，１Ｈ，Ｊ＝２．３Ｈｚ），７．４２（ｄｄ，１Ｈ，Ｊ＝１．１，５．１Ｈｚ），７．３３（ｄｄ，１Ｈ，Ｊ＝１．１，３．５Ｈｚ），７．０６（ｄｄ，１Ｈ，Ｊ＝３．６，５．１Ｈｚ），６．５９（ｔ，１Ｈ，Ｊ＝５．７Ｈｚ），５．３０（ｄ，１Ｈ，Ｊ＝４．６Ｈｚ），５．１７（ｔ，１Ｈ，Ｊ＝５．１Ｈｚ），４．２８（ｍ，Ｈ），３．８６（ｍ，１Ｈ），３．７０−３．７４（ｍ，１Ｈ），３．５８−３．６９（ｍ，１Ｈ），２．４１−２．４５（ｍ，１Ｈ），２．２５−２．３３（ｍ，１Ｈ）．
　３）　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（フラン−２−イル）−２−ニトロピロールの合成
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロール（１７７ｍｇ，０．５ｍｍｏｌ）、二塩化ビス（トリフェニルホスフィン）パラジウム（ＩＩ）（１８ｍｇ，０．０２５ｍｍｏｌ）のＤＭＦ（２．５ｍｌ）溶液に２−（トリブチルスタニル）フラン（４７２μｌ，１．５ｍｍｏｌ）を加えた。マイクロウェーブ装置（スタンダードモード）で１００℃，３０分間反応後、酢酸エチル（５０ｍｌ）、水（５０ｍｌ）で分液し、有機層を濃縮した。これをＨＰＬＣ精製し、１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（フラン−２−イル）−２−ニトロピロール（１１１ｍｇ，０．３８ｍｍｏｌ，７６％）を得た。 Properties of 1- (2-deoxy-β-D-ribofuranosyl) -4- (thien-2-yl) -2-nitropyrrole 1 H NMR (300 MHz, DMSO-d6) δ 8.13 (d, 1H, J = 2.3 Hz), 7.52 (d, 1H, J = 2.3 Hz), 7.42 (dd, 1H, J = 1.1, 5.1 Hz), 7.33 (dd, 1H, J = 1.1, 3.5 Hz), 7.06 (dd, 1H, J = 3.6, 5.1 Hz), 6.59 (t, 1H, J = 5.7 Hz), 5.30 (d, 1H, J = 4.6 Hz), 5.17 (t, 1H, J = 5.1 Hz), 4.28 (m, H), 3.86 (m, 1H), 3.70-3.74 ( m, 1H), 3.58-3.69 (m, 1H), 2.41-2.45 (m, 1H), 2.25-2.33 (m, 1H).
3) Synthesis of 1- (2-deoxy-β-D-ribofuranosyl) -4- (furan-2-yl) -2-nitropyrrole 1- (2-deoxy-β-D-ribofuranosyl) -4-iodo- To a solution of 2-nitropyrrole (177 mg, 0.5 mmol), bis (triphenylphosphine) palladium dichloride (II) (18 mg, 0.025 mmol) in DMF (2.5 ml) was added 2- (tributylstannyl) furan (472 μl). , 1.5 mmol). After reacting at 100 ° C. for 30 minutes in a microwave apparatus (standard mode), the mixture was separated with ethyl acetate (50 ml) and water (50 ml), and the organic layer was concentrated. This was purified by HPLC to obtain 1- (2-deoxy-β-D-ribofuranosyl) -4- (furan-2-yl) -2-nitropyrrole (111 mg, 0.38 mmol, 76%).
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（フラン−２−イル）−２−ニトロピロールの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　８．０８（ｄ，１Ｈ，Ｊ＝２．３Ｈｚ），７．６３（ｄｄ，１Ｈ，Ｊ＝０．７，１．８Ｈｚ），７．５０（ｄ，１Ｈ，Ｊ＝２．３Ｈｚ），６．６９（ｄｄ，１Ｈ，Ｊ＝０．７，３．３Ｈｚ），６．６１（ｔ，１Ｈ，Ｊ＝５．７Ｈｚ），６．５３（ｄｄ，１Ｈ，Ｊ＝１．８，３．３Ｈｚ），５．２９（ｄ，１Ｈ，Ｊ＝４．４Ｈｚ），５．１２（ｔ，１Ｈ，Ｊ＝５．１Ｈｚ），４．２７（ｍ，１Ｈ），３．８７（ｍ，１Ｈ），３．６５−３．７２（ｍ，１Ｈ），３．５６−３．６３（ｍ，１Ｈ），２．４１−２．４６（ｍ，１Ｈ），２．２３−２．３１（ｍ，１Ｈ）．
　４）　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（２，２’−ビチエン−５−イル）−２−ニトロピロールの合成
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロール（１７７ｍｇ，０．５ｍｍｏｌ）、二塩化ビス（トリフェニルホスフィン）パラジウム（ＩＩ）（１８ｍｇ，０．０２５ｍｍｏｌ）のＤＭＦ（２．５ｍｌ）溶液に２−（トリブチルスタニル）ジチオフェン（３４１ｍｇ，０．７５ｍｍｏｌ）を加えた。マイクロウェーブ装置（スタンダードモード）で１００℃，３０分間反応後、酢酸エチル（５０ｍｌ）、水（５０ｍｌ）で分液し、有機層を濃縮した。これをＨＰＬＣ精製し、１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（２，２’−ビチエン−５−イル）−２−ニトロピロール（９０ｍｇ，０．２３ｍｍｏｌ，４６％）を得た。 Properties of 1- (2-deoxy-β-D-ribofuranosyl) -4- (furan-2-yl) -2-nitropyrrole 1 H NMR (300 MHz, DMSO-d6) δ 8.08 (d, 1H, J = 2.3 Hz), 7.63 (dd, 1H, J = 0.7, 1.8 Hz), 7.50 (d, 1H, J = 2.3 Hz), 6.69 (dd, 1H, J = 0.7, 3.3 Hz), 6.61 (t, 1H, J = 5.7 Hz), 6.53 (dd, 1H, J = 1.8, 3.3 Hz), 5.29 (d, 1H, J = 4.4 Hz), 5.12 (t, 1H, J = 5.1 Hz), 4.27 (m, 1H), 3.87 (m, 1H), 3.65-3.72 ( m, 1H), 3.56-3.63 (m, 1H), 2.41-2.46 (m, 1H), 2.23-2.31 (m, 1H).
4) Synthesis of 1- (2-deoxy-β-D-ribofuranosyl) -4- (2,2′-bithien-5-yl) -2-nitropyrrole 1- (2-deoxy-β-D-ribofuranosyl) To a solution of -4-iodo-2-nitropyrrole (177 mg, 0.5 mmol), bis (triphenylphosphine) palladium dichloride (II) (18 mg, 0.025 mmol) in DMF (2.5 ml) was added 2- (tributylsterol). Nyl) dithiophene (341 mg, 0.75 mmol) was added. After reacting at 100 ° C. for 30 minutes in a microwave apparatus (standard mode), the mixture was separated with ethyl acetate (50 ml) and water (50 ml), and the organic layer was concentrated. This was purified by HPLC to give 1- (2-deoxy-β-D-ribofuranosyl) -4- (2,2′-bithien-5-yl) -2-nitropyrrole (90 mg, 0.23 mmol, 46%). Obtained.
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−（２，２’−ビチエン−５−イル）−２−ニトロピロールの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　８．１５（ｄ，１Ｈ，Ｊ＝２．３Ｈｚ），７．５７（ｄ，１Ｈ，Ｊ＝２．３Ｈｚ），７．５０（ｄｄ，１Ｈ，Ｊ＝１．１，５．１Ｈｚ），７．２４−７．３１（ｍ，３Ｈ），７．０８（ｄｄ，１Ｈ，Ｊ＝３．６，５．１Ｈｚ），６．６０（ｔ，１Ｈ，Ｊ＝５．７Ｈｚ），５．２８（ｄ，１Ｈ，Ｊ＝３．６Ｈｚ），５．１７（ｔ，１Ｈ，Ｊ＝５．２Ｈｚ），４．２９（ｍ，１Ｈ），３．８７（ｍ，１Ｈ），３．６８−３．７５（ｍ，１Ｈ），３．５７−３．６５（ｍ，１Ｈ），２．４１−２．４６（ｍ，１Ｈ），２．２６−２．３４（ｍ，１Ｈ）．
　５）　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−メチル−２−ニトロピロールの合成
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロール（１４２ｍｇ，０．４ｍｍｏｌ）、二塩化ビス（トリフェニルホスフィン）パラジウム（ＩＩ）（１４ｍｇ，０．０２ｍｍｏｌ）、トリフェニルアルシン（１２ｍｇ，０．０４ｍｍｏｌ）のＤＭＦ（２ｍｌ）溶液にテトラメチルスズ（２８７μｌ，２ｍｍｏｌ）を加えた。６０℃，２日間反応後、酢酸エチル（５０ｍｌ）、水（５０ｍｌ）で分液し、有機層を濃縮した。これをＨＰＬＣ精製し、１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−メチル−２−ニトロピロール（１５ｍｇ，０．０６ｍｍｏｌ，１５％）を得た。 1- (2-deoxy-β-D-ribofuranosyl) -4- (2,2′-bithien-5-yl) -2-nitropyrrole property value 1 H NMR (300 MHz, DMSO-d6) δ 8.15 (D, 1H, J = 2.3 Hz), 7.57 (d, 1H, J = 2.3 Hz), 7.50 (dd, 1H, J = 1.1, 5.1 Hz), 7.24- 7.31 (m, 3H), 7.08 (dd, 1H, J = 3.6, 5.1 Hz), 6.60 (t, 1H, J = 5.7 Hz), 5.28 (d, 1H) , J = 3.6 Hz), 5.17 (t, 1H, J = 5.2 Hz), 4.29 (m, 1H), 3.87 (m, 1H), 3.68-3.75 (m , 1H), 3.57-3.65 (m, 1H), 2.41-2.46 (m, 1H), 2.26-2.34 (m, 1H).
5) Synthesis of 1- (2-deoxy-β-D-ribofuranosyl) -4-methyl-2-nitropyrrole 1- (2-deoxy-β-D-ribofuranosyl) -4-iodo-2-nitropyrrole (142 mg , 0.4 mmol), bis (triphenylphosphine) palladium dichloride (II) (14 mg, 0.02 mmol), trimethylarsine (12 mg, 0.04 mmol) in DMF (2 ml) solution with tetramethyltin (287 μl, 2 mmol) ) Was added. After reacting at 60 ° C. for 2 days, the mixture was partitioned between ethyl acetate (50 ml) and water (50 ml), and the organic layer was concentrated. This was purified by HPLC to obtain 1- (2-deoxy-β-D-ribofuranosyl) -4-methyl-2-nitropyrrole (15 mg, 0.06 mmol, 15%).
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−メチル−２−ニトロピロールの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　７．５５（ｄ，１Ｈ，Ｊ＝２．８Ｈｚ），７．０９（ｄ，１Ｈ，Ｊ＝２．２Ｈｚ），６．５５（ｔ，１Ｈ，Ｊ＝５．９Ｈｚ），５．２７（ｄ，１Ｈ，Ｊ＝４．３Ｈｚ），５．００（ｔ，１Ｈ，Ｊ＝５．３Ｈｚ），４．２２（ｍ，１Ｈ），３．８２（ｍ，１Ｈ），３．５２−３．６４（ｍ，２Ｈ），２．３４−２．４２（ｍ，１Ｈ），２．１１−２．１９（ｍ，１Ｈ），２．０２（ｓ，３Ｈ）．
　６）　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−プロピニル−２−ニトロピロールの合成（図３、Ｒ＝−ＣＨ３）
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−ヨード−２−ニトロピロール（１８０ｍｇ，０．５ｍｍｏｌ）、二塩化ビス（トリフェニルホスフィン）パラジウム（ＩＩ）（３８ｍｇ，０．０５ｍｍｏｌ）のＤＭＦ（５ｍｌ）溶液にトリブチル（１−プロピニル）スズ（３２７μｌ，１ｍｍｏｌ）を加えた。１００　℃，　９０分間反応後、濃縮した。これをシリカゲルカラムクロマトグラフィーおよびＨＰＬＣ精製し、１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−プロピニル−２−ニトロピロール（７６ｍｇ，０．２８ｍｍｏｌ，５７％）を得た。 Properties of 1- (2-deoxy-β-D-ribofuranosyl) -4-methyl-2-nitropyrrole 1 H NMR (300 MHz, DMSO-d6) δ 7.55 (d, 1H, J = 2.8 Hz) 7.09 (d, 1H, J = 2.2 Hz), 6.55 (t, 1H, J = 5.9 Hz), 5.27 (d, 1H, J = 4.3 Hz), 5.00 ( t, 1H, J = 5.3 Hz), 4.22 (m, 1H), 3.82 (m, 1H), 3.52-3.64 (m, 2H), 2.34-2.42 ( m, 1H), 2.11-2.19 (m, 1H), 2.02 (s, 3H).
6) Synthesis of 1- (2-deoxy-β-D-ribofuranosyl) -4-propynyl-2-nitropyrrole (FIG. 3, R = —CH 3 )
1- (2-deoxy-β-D-ribofuranosyl) -4-iodo-2-nitropyrrole (180 mg, 0.5 mmol), bis (triphenylphosphine) palladium (II) dichloride (38 mg, 0.05 mmol) Tributyl (1-propynyl) tin (327 μl, 1 mmol) was added to a DMF (5 ml) solution. After reacting at 100 ° C. for 90 minutes, the mixture was concentrated. This was purified by silica gel column chromatography and HPLC to give 1- (2-deoxy-β-D-ribofuranosyl) -4-propynyl-2-nitropyrrole (76 mg, 0.28 mmol, 57%).
　１−（２−デオキシ−β−Ｄ−リボフラノシル）−４−プロピニル−２−ニトロピロールの物性値
　１Ｈ　ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ６）　δ　７．９２（ｄ，１Ｈ，Ｊ＝２．２Ｈｚ），７．２７（ｄ，１Ｈ，Ｊ＝２．２Ｈｚ），６．５５（ｔ，１Ｈ，Ｊ＝５．７Ｈｚ），５．２８（ｄ，１Ｈ，Ｊ＝４．５Ｈｚ），５．１１（ｔ，１Ｈ，Ｊ＝５．２Ｈｚ），４．２４（ｍ，１Ｈ），３．８５（ｍ，１Ｈ），３．５３−３．７０（ｍ，２Ｈ），２．４５（ｍ，１Ｈ），２．２２（ｍ，１Ｈ），１．９９（ｓ，３Ｈ）．
 1- (2-deoxy-β-D-ribofuranosyl) -4-propynyl-2-nitropyrrole property value 1 H NMR (300 MHz, DMSO-d6) δ 7.92 (d, 1H, J = 2.2 Hz) 7.27 (d, 1H, J = 2.2 Hz), 6.55 (t, 1H, J = 5.7 Hz), 5.28 (d, 1H, J = 4.5 Hz), 5.11 ( t, 1H, J = 5.2 Hz), 4.24 (m, 1H), 3.85 (m, 1H), 3.53-3.70 (m, 2H), 2.45 (m, 1H) 2.22 (m, 1H), 1.99 (s, 3H).

Claims (20)

  1. 　式Ｉで示す２−ニトロピロール構造を有する消光剤
    Figure JPOXMLDOC01-appb-C000001
     ［式Ｉにおいて、Ｒ１及びＲ２は
    　リボース、デオキシリボース、
    　水素、水酸基、ＳＨ基、ハロゲン、
    　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
    　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
    　糖、糖鎖、アミノ酸、ペプチド、
    　リンカーを介して結合した蛍光性分子
    からなる群から独立に選択される基である］。     Quenching agent having 2-nitropyrrole structure represented by formula I
    Figure JPOXMLDOC01-appb-C000001
     [In Formula I, R 1 and R 2 are ribose, deoxyribose,
    Hydrogen, hydroxyl group, SH group, halogen,
    A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
    One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
    Sugar, sugar chain, amino acid, peptide,
    A group independently selected from the group consisting of fluorescent molecules linked via a linker.
  2. 　式Ｉにおいて、Ｒ１がリボースまたはデオキシリボースである、請求項１に記載の消光剤。 The quencher according to claim 1, wherein in formula I, R 1 is ribose or deoxyribose.
  3. 　人工塩基対の形成を検出する方法であって、
    　１）式ＩＩ
    Figure JPOXMLDOC01-appb-C000002
     ［式ＩＩにおいて、Ｒ２は、
    　水素、水酸基、ＳＨ基、ハロゲン、
    　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
    　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
    　糖、糖鎖、アミノ酸、ペプチド、
    　リンカーを介して結合した蛍光性分子、
    からなる群から選択される基である］
    で表される消光性人工塩基を有するヌクレオシドまたはヌクレオチド、あるいは、
    　２）蛍光共鳴エネルギー転移（ＦＲＥＴ）又は静的消光作用の供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するヌクレオシドまたはヌクレオチド
    のいずれか、あるいは双方を用いることを特徴とする、前記方法。     A method for detecting the formation of an artificial base pair,
    1) Formula II
    Figure JPOXMLDOC01-appb-C000002
     [In Formula II, R 2 is
    Hydrogen, hydroxyl group, SH group, halogen,
    A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
    One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
    Sugar, sugar chain, amino acid, peptide,
    A fluorescent molecule bound via a linker,
    A group selected from the group consisting of]
    A nucleoside or nucleotide having a quenching artificial base represented by:
    2) Use of a natural base modifier, an artificial base, or a nucleoside or nucleotide having a base analog having self-quenching which can be a donor of fluorescence resonance energy transfer (FRET) or static quenching action, or both. Characterized by the above.
  4. 　人工塩基の塩基対の形成を検出する方法であって、蛍光性人工塩基と式ＩＩ
    Figure JPOXMLDOC01-appb-C000003
     ［式ＩＩにおいて、Ｒ２は、
    　水素、水酸基、ＳＨ基、ハロゲン、
    　置換又は未置換の、炭素数２ないし１０のアルキル基、アルケニル基又はアルキニル基、
    　窒素原子または硫黄原子を含む、１又は複数の５員ヘテロ環、１又は複数の６員ヘテロ環、１又は複数の複素環ヘテロ環、１又は複数の芳香族環、
    　糖、糖鎖、アミノ酸、ペプチド、
    　リンカーを介して結合した蛍光性分子、
    からなる群から選択される基である］
    で表される消光性人工塩基との塩基対形成により、蛍光性人工塩基の蛍光の低下を観察することにより、人工塩基対が形成されたことが検出される、前記方法。     A method for detecting the formation of a base pair of an artificial base comprising a fluorescent artificial base and formula II
    Figure JPOXMLDOC01-appb-C000003
     [In Formula II, R 2 is
    Hydrogen, hydroxyl group, SH group, halogen,
    A substituted or unsubstituted alkyl group having 2 to 10 carbon atoms, an alkenyl group or an alkynyl group,
    One or more 5-membered heterocycles, one or more 6-membered heterocycles, one or more heterocycles, one or more aromatic rings, including nitrogen or sulfur atoms,
    Sugar, sugar chain, amino acid, peptide,
    A fluorescent molecule bound via a linker,
    A group selected from the group consisting of]
    The method as described above, wherein the formation of an artificial base pair is detected by observing a decrease in fluorescence of the fluorescent artificial base by forming a base pair with the quenching artificial base represented by formula (1).
  5. 　蛍光性人工塩基の蛍光の低下により、人工塩基の塩基対の形成を検出する方法であって、以下の
    　（ｉ）７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）；
    　（ｉｉ）７−（２，２’，５’，２’’−ターチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓｓ）；
    　（ｉｉｉ）２−アミノ−６−（２，２’−ビチエン−５−イル）プリン−９−イル基（ｓｓ）；
    　（ｉｖ）２−アミノ−６−（２，２’，５’，２’’−ターチエン−５−イル）プリン−９−イル基（ｓｓｓ）；
    　（ｖ）４−（２，２’−ビチエン−５−イル）−ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｓ）；
    　（ｖｉ）４−［２−（２−チアゾリル）チエン−５−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｓａｖ）；及び
    　（ｖｉｉ）４−［５−（２−チエニル）チアゾール−２−イル］ピロロ［２，３−ｂ］ピリジン−１−イル基（Ｄｖａｓ）；
    からなる群より選択される蛍光性人工塩基と、
    　以下の式ＩＩＩ，又は式ＩＶの消光性塩基
    Figure JPOXMLDOC01-appb-C000004
     ［式ＩＩＩにおいて、Ｒ３は、
    　−Ｈ、ヨード、−ＣＨ３、
    Figure JPOXMLDOC01-appb-C000005
     から選択される］
    Figure JPOXMLDOC01-appb-C000006
     ［式ＩＶにおいて、Ｒ４は、
    　−ＣＨ３、−ＣＨ２−ＮＨ２、及び
    Figure JPOXMLDOC01-appb-C000007
     （ここにおいて、ｎは０ないし１２のいずれかの整数である）
    から選択される］
    との間で塩基対が形成されると、蛍光性人工塩基の蛍光が低下し、人工塩基対が形成されたことが検出される、前記方法。     A method for detecting the formation of a base pair of an artificial base by lowering the fluorescence of a fluorescent artificial base, comprising the following (i) 7- (2,2′-bithien-5-yl) imidazo [4,5- b] Pyridin-3-yl group (Dss);
    (Ii) 7- (2,2 ′, 5 ′, 2 ″ -tertien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dsss);
    (Iii) 2-amino-6- (2,2′-bithien-5-yl) purin-9-yl group (ss);
    (Iv) 2-amino-6- (2,2 ′, 5 ′, 2 ″ -tert-en-5-yl) purin-9-yl group (sss);
    (V) 4- (2,2′-bithien-5-yl) -pyrrolo [2,3-b] pyridin-1-yl group (Dsas);
    (Vi) 4- [2- (2-thiazolyl) thien-5-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dsav); and (vii) 4- [5- (2-thienyl) ) Thiazol-2-yl] pyrrolo [2,3-b] pyridin-1-yl group (Dvas);
    A fluorescent artificial base selected from the group consisting of:
    Quenching bases of the following formula III or formula IV
    Figure JPOXMLDOC01-appb-C000004
     [In Formula III, R 3 is
    -H, iodine, -CH 3,
    Figure JPOXMLDOC01-appb-C000005
     Selected from]
    Figure JPOXMLDOC01-appb-C000006
     [In Formula IV, R 4 is
    -CH 3, -CH 2 -NH 2 and,
    Figure JPOXMLDOC01-appb-C000007
     (Where n is an integer from 0 to 12)
    Selected from]
    When the base pair is formed between the fluorescent artificial base, the fluorescence of the fluorescent artificial base decreases, and it is detected that the artificial base pair is formed.
  6. 　７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）を塩基として有するポリヌクレオチドを含む、核酸プライマー；並びに、
    　以下の式ＩＩＩ，又は式ＩＶの消光性塩基
    Figure JPOXMLDOC01-appb-C000008
     ［式ＩＩＩにおいて、Ｒ３は、
    　−Ｈ、ヨード、−ＣＨ３、
    Figure JPOXMLDOC01-appb-C000009
     から選択される］
    Figure JPOXMLDOC01-appb-C000010
     ［式ＩＶにおいて、Ｒ４は、
    　−ＣＨ３、−ＣＨ２−ＮＨ２、及び
    Figure JPOXMLDOC01-appb-C000011
     （ここにおいて、ｎは０ないし１２のいずれかの整数である）
    から選択される］
    を塩基として有するポリヌクレオチド
    を含む、蛍光性人工塩基の蛍光の低下により、人工塩基の塩基対の形成を検出する方法に使用するためのキット。     A nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base; and
    Quenching bases of the following formula III or formula IV
    Figure JPOXMLDOC01-appb-C000008
     [In Formula III, R 3 is
    -H, iodine, -CH 3,
    Figure JPOXMLDOC01-appb-C000009
     Selected from]
    Figure JPOXMLDOC01-appb-C000010
     [In Formula IV, R 4 is
    -CH 3, -CH 2 -NH 2 and,
    Figure JPOXMLDOC01-appb-C000011
     (Where n is an integer from 0 to 12)
    Selected from]
    A kit for use in a method for detecting the formation of a base pair of an artificial base by reducing the fluorescence of the fluorescent artificial base, comprising a polynucleotide having a base as a base.
  7. 　人工塩基対を検出する方法であって、
    　式Ｖ
    Figure JPOXMLDOC01-appb-C000012
     ［式Ｖにおいて、Ｒ５は、リンカーを介して結合した蛍光性分子である］
    で表される消光性人工塩基中の蛍光性分子の蛍光強度が、式Ｖの人工塩基が塩基対を形成することによって変化し、人工塩基対が形成されたことが検出される、前記方法。     A method for detecting artificial base pairs,
    Formula V
    Figure JPOXMLDOC01-appb-C000012
     [In Formula V, R 5 is a fluorescent molecule bound via a linker]
    The method, wherein the fluorescence intensity of the fluorescent molecule in the quenching artificial base represented by formula (1) is changed by the formation of the base pair by the artificial base of Formula V, and the formation of the artificial base pair is detected.
  8. 　蛍光強度の変化により、人工塩基の塩基対の形成を検出する方法であって、
    　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、以下の式ＶＩの塩基：
    Figure JPOXMLDOC01-appb-C000013
     ［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
    　式ＶＩの塩基中の蛍光性分子の蛍光強度が増加し、人工塩基対が形成されたことが検出される、前記方法。     A method for detecting the formation of a base pair of an artificial base by a change in fluorescence intensity,
    7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
    Figure JPOXMLDOC01-appb-C000013
     In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
    Said method wherein the fluorescence intensity of the fluorescent molecule in the base of formula VI is increased and it is detected that an artificial base pair has been formed.
  9. 　蛍光性分子が、インドカルボシアニン（Ｃｙ３）、インドジカルボシアニン（Ｃｙ５）、５−カルボキシフルオレセイン（５−ＦＡＭ）、６−カルボキシフルオレセイン（６−ＦＡＭ）、５−カルボキシテトラメチルローダミン（５−ＴＡＭＲＡ）、６−カルボキシテトラメチルローダミン（６−ＴＡＭＲＡ）、５−ジメチルアミノナフタレン−１−スルホン酸（ＤＡＮＳＹＬ）、５−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（５−ＨＥＸ）、６−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（６−ＨＥＸ）、５−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（５−ＴＥＴ）、６−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（６−ＴＥＴ）、５−カルボキシ−Ｘ−ローダミン（５−ＲＯＸ）、及び６−カルボキシ−Ｘ−ローダミン（６−ＲＯＸ）からなる群から選択される、請求項７又は８に記載の方法。 Fluorescent molecules include indocarbocyanine (Cy3), indodicarbocyanine (Cy5), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA) ), 6-carboxytetramethylrhodamine (6-TAMRA), 5-dimethylaminonaphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (5-HEX), 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (6-HEX), 5-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (5-TET), 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein ( -Tet), 5-carboxy -X- rhodamine (5-ROX), and is selected from the group consisting of 6-carboxy -X- rhodamine (6-ROX), The method according to claim 7 or 8.
  10. 　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）を塩基として有するポリヌクレオシドを含む、核酸プライマー、並びに、
    　式ＶＩの塩基：
    Figure JPOXMLDOC01-appb-C000014
     ［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］を塩基として有するポリヌクレオチド
    を含む、蛍光強度の変化により、人工塩基の塩基対の形成を検出する方法に使用するためのキット。     A nucleic acid primer comprising a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base; and
    Base of formula VI:
    Figure JPOXMLDOC01-appb-C000014
     [In Formula VI, R 6 is a fluorescent molecule bound through or without a linker], and detects the formation of an artificial base base pair by a change in fluorescence intensity. Kit for use in the method.
  11. 　人工塩基対の形成を検出する方法であって、
    　蛍光共鳴エネルギー転移（ＦＲＥＴ）又は静的消光作用の供与体となりうる自己消光性を有する天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドを含む核酸を利用し、当該核酸中の人工塩基（第一人工塩基）と蛍光性分子を有する人工塩基（第二人工塩基）との間で人工塩基対が形成されると、前記天然塩基修飾体、人工塩基、或いは塩基類似体を有するポリヌクレオシドから、第二人工塩基の有する蛍光性分子への蛍光共鳴エネルギー転移又は静的消光作用が生じて、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、前記方法。     A method for detecting the formation of an artificial base pair,
    Using a nucleic acid comprising a natural base modification, an artificial base or a base nucleoside having a base analog having self-quenching which can serve as a donor for fluorescence resonance energy transfer (FRET) or static quenching action, When an artificial base pair is formed between a base (first artificial base) and an artificial base having a fluorescent molecule (second artificial base), the natural base modifier, artificial base, or base analog The method, wherein a fluorescence resonance energy transfer or a static quenching action from a nucleoside to a fluorescent molecule of a second artificial base occurs to detect that a fluorescence spectrum is changed and an artificial base pair is formed.
  12. 　蛍光共鳴エネルギー転移又は静的消光作用による蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
    　７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）と、以下の式ＶＩの塩基：
    Figure JPOXMLDOC01-appb-C000015
     ［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
    　２４０−４１０ｎｍの紫外線による励起により、Ｄｓｓから式ＶＩの塩基中の蛍光性分子
    への蛍光共鳴エネルギー転移又は静的消光作用が生じて、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、前記方法。     A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum caused by fluorescence resonance energy transfer or static quenching,
    7- (2,2′-Bitien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) and a base of formula VI below:
    Figure JPOXMLDOC01-appb-C000015
     In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
    Excitation with ultraviolet light at 240-410 nm caused fluorescence resonance energy transfer or static quenching action from Dss to the fluorescent molecule in the base of formula VI, resulting in a change in the fluorescence spectrum and the formation of artificial base pairs. Said method being detected.
  13. 　蛍光共鳴エネルギー転移又は静的消光作用による蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
    　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、以下の式ＶＩの塩基：
    Figure JPOXMLDOC01-appb-C000016
     ［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
    　２４０−３９０ｎｍの紫外線による励起により、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移又は静的消光作用が生じ、蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
    　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）を塩基として有するポリヌクレオチドが少なくとも１つ存在する、前記方法。     A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum caused by fluorescence resonance energy transfer or static quenching,
    7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
    Figure JPOXMLDOC01-appb-C000016
     In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
    Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI by excitation with UV light at 240-390 nm A static quenching effect occurs, the fluorescence spectrum changes and the formation of artificial base pairs is detected,
    Here, at least one polynucleotide having a 2-amino-6- (2-thienyl) purin-9-yl group (s) as a base is present on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. Said method.
  14. 　蛍光共鳴エネルギー転移又は静的消光作用による蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
    　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓ）と、以下の式ＶＩの塩基：
    Figure JPOXMLDOC01-appb-C000017
     ［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
    　３５０−３９０ｎｍの紫外線による励起により、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移又は静的消光作用が生じて蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
    　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、少なくとも１つの２−アミノ−６−（２−チエニル）プリン−９−イル基（ｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドが少なくとも１つ存在する、
    前記方法     A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum caused by fluorescence resonance energy transfer or static quenching,
    7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
    Figure JPOXMLDOC01-appb-C000017
     In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
    Fluorescence resonance energy transfer from at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) to a fluorescent molecule in a base of formula VI by excitation with ultraviolet light at 350-390 nm A static quenching action occurs, the fluorescence spectrum changes, and it is detected that an artificial base pair is formed,
    Here, a base in which at least one 2-amino-6- (2-thienyl) purin-9-yl group (s) is bound to a natural base on the same strand as a nucleic acid containing a polynucleoside having Ds as a base. There is at least one polynucleotide having
    Said method
  15. 　蛍光共鳴エネルギー転移又は静的消光作用による蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法であって、
    　７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）と、以下の式ＶＩの塩基：
    Figure JPOXMLDOC01-appb-C000018
     ［式ＶＩにおいて、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］との間で塩基対が形成されると、
    　２４０−４１０ｎｍの紫外線による励起により、７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）から、式ＶＩの塩基中の蛍光性分子への蛍光共鳴エネルギー転移又は静的消光作用が生じて蛍光スペクトルが変化し、人工塩基対が形成されたことが検出される、
    　ここにおいて、Ｄｓを塩基として有するポリヌクレオシドを含む核酸と同一鎖上に、少
    なくとも１つの７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドが存在する、
    前記方法。     A method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum caused by fluorescence resonance energy transfer or static quenching,
    7- (2-Thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) and a base of formula VI below:
    Figure JPOXMLDOC01-appb-C000018
     In Formula VI, when R 6 is a fluorescent molecule bound through or without a linker, a base pair is formed,
    Fluorescence in the base of formula VI from 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) upon excitation with 240-410 nm UV light Fluorescence resonance energy transfer or static quenching action to the molecule occurs to change the fluorescence spectrum and detect the formation of artificial base pairs.
    Here, at least one 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group on the same strand as the nucleic acid containing a polynucleoside having Ds as a base There is a polynucleotide having a base in which (Dss) is bound to a natural base,
    Said method.
  16. 　蛍光性物質が、インドカルボシアニン（Ｃｙ３）、インドジカルボシアニン（Ｃｙ５）、５−カルボキシフルオレセイン（５−ＦＡＭ）、６−カルボキシフルオレセイン（６−ＦＡＭ）、５−カルボキシテトラメチルローダミン（５−ＴＡＭＲＡ）、６−カルボキシテトラメチルローダミン（６−ＴＡＭＲＡ）、５−ジメチルアミノナフタレン−１−スルホン酸（ＤＡＮＳＹＬ）、５−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（５−ＨＥＸ）、６−カルボキシ−２’，４，４’，５’，７，７’−ヘキサクロロフルオレセイン（６−ＨＥＸ）、５−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（５−ＴＥＴ）、６−カルボキシ−２’，４，７，７’−テトラクロロフルオレセイン（６−ＴＥＴ）、５−カルボキシ−Ｘ−ローダミン（５−ＲＯＸ）、及び６−カルボキシ−Ｘ−ローダミン（６−ＲＯＸ）からなる群から選択される、請求項１１ないし１５のいずれか１項に記載の方法。 Fluorescent substances include indocarbocyanine (Cy3), indodicarbocyanine (Cy5), 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), 5-carboxytetramethylrhodamine (5-TAMRA) ), 6-carboxytetramethylrhodamine (6-TAMRA), 5-dimethylaminonaphthalene-1-sulfonic acid (DANSYL), 5-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (5-HEX), 6-carboxy-2 ′, 4,4 ′, 5 ′, 7,7′-hexachlorofluorescein (6-HEX), 5-carboxy-2 ′, 4,7,7′-tetrachloro Fluorescein (5-TET), 6-carboxy-2 ′, 4,7,7′-tetrachlorofluorescein ( 16. -TET), 5-carboxy-X-rhodamine (5-ROX), and 6-carboxy-X-rhodamine (6-ROX). the method of.
  17. 　式ＶＩの塩基中の置換基Ｒ６が以下の：
    Figure JPOXMLDOC01-appb-C000019
     の構造を有する、請求項１２−１５に記載の方法。     The substituent R 6 in the base of formula VI is:
    Figure JPOXMLDOC01-appb-C000019
     The method according to claim 12-15, which has the structure:
  18. 　検出スペクトルの変化が、肉眼で判定できる、請求項１１ないし１７のいずれか１項に記載の方法。 The method according to any one of claims 11 to 17, wherein a change in the detection spectrum can be determined with the naked eye.
  19. 　核酸の塩基対が、転写、逆転写、複製又は翻訳の工程で形成される、請求項１１ないし１８のいずれか１項に記載の方法。 The method according to any one of claims 11 to 18, wherein the nucleic acid base pair is formed by a step of transcription, reverse transcription, replication or translation.
  20. 　以下のｉ）−ｉｉｉ）からなる群から選択される、１つの核酸プライマー；
    　ｉ）７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）を塩基として有するポリヌクレオチドを含む、核酸プライマー；
    　ｉｉ）７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）を塩基として有するポリヌクレオシド、及び、少なくとも１つの、２−アミノ−６−（２−チエニル）−９Ｈ−プリン−９−イル基（ｓ）を塩基として有するポリヌクレオチドを含む、核酸プライマー；
    　ｉｉｉ）７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓ）を塩基として有するポリヌクレオシド、及び、少なくとも１つの、２−アミノ−６−（２−チエニル）−９Ｈ−プリン−９−イル基（ｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドを含む、核酸プライマー；及び
    　ｉｖ）７−（２−チエニル）イミダゾ［４，５−ｂ］ピリジン３−イル基（Ｄｓ）を塩基として有するポリヌクレオシド、及び、７−（２，２’−ビチエン−５−イル）イミダゾ［４，５−ｂ］ピリジン−３−イル基（Ｄｓｓ）が天然型塩基に結合した塩基を有するポリヌクレオチドを含む、核酸プライマー、
    　並びに、式ＶＩの塩基：
    Figure JPOXMLDOC01-appb-C000020
     ［式ＶＩにおいてここで、Ｒ６は、リンカーを介して又は介さずに結合した蛍光性分子である］
    を塩基として有するポリヌクレオチド
    を含む、蛍光共鳴エネルギー転移又は静的消光作用による蛍光スペクトルの変化により、人工塩基の塩基対の形成を検出する方法に使用するためのキット。     One nucleic acid primer selected from the group consisting of i) -iii) below:
    i) a nucleic acid primer comprising a polynucleotide having 7- (2,2′-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) as a base;
    ii) a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base, and at least one 2-amino-6- (2-thienyl)- A nucleic acid primer comprising a polynucleotide having a 9H-purin-9-yl group (s) as a base;
    iii) a polynucleoside having a 7- (2-thienyl) imidazo [4,5-b] pyridin-3-yl group (Ds) as a base, and at least one 2-amino-6- (2-thienyl) A nucleic acid primer comprising a polynucleotide having a base to which a -9H-purin-9-yl group (s) is bound to a natural base; and iv) 7- (2-thienyl) imidazo [4,5-b] pyridine 3 -A polynucleoside having an yl group (Ds) as a base and a 7- (2,2'-bithien-5-yl) imidazo [4,5-b] pyridin-3-yl group (Dss) is a natural base A nucleic acid primer comprising a polynucleotide having a base bound to
    And a base of formula VI:
    Figure JPOXMLDOC01-appb-C000020
     [Wherein R 6 is a fluorescent molecule attached via or without a linker in Formula VI]
    A kit for use in a method for detecting the formation of a base pair of an artificial base by a change in a fluorescence spectrum by fluorescence resonance energy transfer or static quenching action, comprising a polynucleotide having a base as a base.
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JPWO2014038561A1 (en) * 2012-09-04 2016-08-12 株式会社ダナフォーム Compound, nucleic acid, labeling substance and detection method
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